Agradecemos a la Rata de Recursos y Centro de Investigación de la Universidad de Missouri, Columbia, Missouri, para proporcionar ratas GFP; Neuralstem Inc. para proporcionar células madre neurales humanas. Este trabajo fue apoyado por la Administración de Veteranos, el NIH (NS09881), la Organización Canadiense de Investigación espinal, la Fundación Craig H. Neilsen, y Bernard y Anne Spitzer Charitable Trust.

Todos los animales protocolos sean aprobados por VA-San Diego Institucional Cuidado de Animales y el empleo Comisión (IACUC). Directrices del NIH para el cuidado de animales de laboratorio y seguridad se siguen estrictamente. Los animales tienen acceso libre a comida y agua durante todo el estudio y se tratan de forma adecuada para reducir al mínimo el dolor y el malestar. 1. Preparación de fibrina componentes que contiene el factor de crecimiento Cocktails <ol><li> Disolver 25 mg de fibrinógeno de rata en 0,5 ml de PBS para obtener 50 mg / ml de solución madre 2x (ver tabla de Materiales). El fibrinógeno es difícil de disolver y se debe colocar en un 37 ° C incubadora o baño de agua y se agita cada 5-10 minutos durante 1-2 horas. </li><li> Disolver 100 trombina rata de la ONU en 2 ml de 10 mM estéril de CaCl2 para obtener 50 U ml 2x solución / acción. </li><li> Disolver los factores de crecimiento en PBS a las siguientes concentraciones para obtener una solución de 100 l existencia: 1 mg / ml: BDNF; NT-3 (alta concentración de valores como la infusión intratecal in vivo <sup&gt; 19); 200 g / ml: GDNF; IGF; bFGF; EGF; PDGF; aFGF; HGF (aproximadamente 1.000 veces superior a la utilizada para el cultivo de células). </li><li> Disolver 25 mg MDL28170 (inhibidor de la calpaína) en 1,3 ml de DMSO para obtener 50 mM solución madre de DMSO y luego se diluye 50 veces en PBS para obtener 1 mM solución madre. </li><li> Mezclar 100 l de cada componente de factor de crecimiento y 100 l de MDL28170 (solución madre 1 mM) para producir 1000 l solución de factor de crecimiento de cóctel (Figura 1). </li><li> Mezclar 500 solución de fibrinógeno l 2x con 500 l de crecimiento factor de cóctel para obtener una solución de 25 mg / ml de fibrinógeno de trabajo que contiene factores de crecimiento y alícuota en 10 o 20 l para el almacenamiento a -70 ° C. La solución es estable durante un máximo de 12 meses a -70 ° C. </li><li> Del mismo modo, mezclar 500 l de trombina solución 2x ​​acción con 500 l de cóctel del factor de crecimiento para obtener la solución de trabajo 25 U / ml de trombina que contiene factores de crecimiento y alícuota en volúmenes de 10 o 20 l similares para el almacenamiento de unt -70 ° C durante un máximo de 12 meses. </li><li> En el día del injerto, colocar cócteles de fibrinógeno y del factor de crecimiento que contiene trombina en hielo hasta que se mezcla con las células. </li></ol> 2. T3 Spinal Cord transección <ol><li> Anestesie hembra adulta Fischer 344 ratas o ratones atímicos usando métodos aceptables. Los sujetos son profundamente anestesiados, cuando la capacidad de respuesta a la cola y la pata pizca están completamente ausentes. Nota: Utilizamos típicamente 150-200 g ratas Fischer o 180-220 g ratas atímicos, y un cóctel de anestesia (2 ml / kg) de ketamina (25 mg / ml), xilazina (1,3 mg / ml) y acepromazina (0,25 mg / ml). </li><li> Aplicar pomada para los ojos para evitar la sequedad o lesiones de los ojos de los animales mientras están bajo anestesia. </li><li> Afeitar el área torácica superior y limpie la piel con Betadine. </li><li> Cortar la piel y el músculo con el n º 15 blade, exponer vértebra T3 usando un retractor quirúrgico, y realizar una laminectomía en T3 vértebras para exponer T3 / 4 de la médula espinal usando una gubia. </li><li> Hacer una lonincisión longitudinal en la duramadre de aproximadamente 2 mm de largo con una cuchilla # 11. </li><li> Coloque iridectomía tijeras debajo de la duramadre para cortar el lateral derecho de toda la médula espinal. Haga otro corte en el mismo lado 1,5 mm más cadually. </li><li> Aspirar el tejido de la médula espinal entre los dos segmentos de corte con un objeto contundente 23 G aguja conectada a vacío intensidad moderada. Utilice la verificación visual para garantizar la transección completa ventral y lateral. Esto completará una hemisección lateral. </li><li> Para extender la lesión a una transección de la médula espinal de ancho completo, coloque incisiones espejo en el hemicord izquierda. Intento de salir de la duramadre intacta, que no sea el primer corte, para retener la matriz de injerto / fibrina firme en el sitio de la lesión cuando se injertan dos semanas más tarde. </li><li> Sutura del músculo, aplique polvo antibiótico y de primera necesidad de la piel. </li><li> Inyectar lactato (3 ml) de Ringer, Bannamine (2,5-5 mg / kg) y ampicilina (80-100 mg / kg) (véase el cuadro Materiales) inmediatamente después de la cirugía y mairatas ntain en una incubadora caliente hasta que se restablezca la termorregulación. </li><li> Vacíe la vejiga e inyectar la solución de Ringer y Ampicilina dos veces al día durante dos semanas, hasta el establecimiento de la vejiga reflejo de vaciado (Figura 1B). </li></ol> 3. Preparación de recién disociada día embrionario 14 Médula Espinal Neural Stem Cells <ol><li> Obtener puras de maíz transgénicos ratas F344 GFP (F344-Tg (UBC-EGFP) F455Rrrc) de Recursos rata y el Centro de Investigación, Universidad de Missouri. </li><li> Inyectar 40 mg des-Gly 10, [D-Ala 6]-hormona luteinizante liberadora de hormona de etilamida (LHRH) diluido en PBS a una concentración de 200 mg / ml, por vía intraperitoneal (IP) por rata hembra madura (8 semanas de edad o más) después de 2 hr -3 inducción luz para la sincronización de recogida de embriones. </li><li> Aparearse con un maduro GFP homocigotos o heterocigotos noche rata macho de 4 días después de la inyección. </li><li> Recoger los embriones en día 13,5-14,5 postcoitus(PC) en tampón HBSS frío, examinar la expresión de GFP bajo unas gafas de sol &quot;NightSea&quot; linterna y filtros (BLS1 - BlueStar Linterna y Modelo VG1 filtro barrera) o un microscopio de fluorescencia. </li><li> Diseccionar la médula espinal a cabo asépticamente de cada feto GFP-positivas y eliminar las meninges que recubren y ganglios espinales adjunto con tijeras de iridectomía y joyeros pinzas finas. </li><li> Recoge las médulas espinales diseccionadas en 2 ml de tampón HBSS frío en un tubo de 15 ml cornical y mantener en hielo. </li><li> Siga referencia # 20 de disociar de médulas espinales diseccionadas: <ol><li> Brevemente, añadir 2 ml de 0,25% de tripsina-EDTA al tubo que contiene de médulas espinales diseccionadas (varios cables pueden ser digeridos juntos, 1 cable puede generar suficientes células para injerto de un sujeto) y digerir a 37 ° C durante 10-12 min. </li><li> Detener la reacción con tripsina por la adición de 10 ml de DMEM que contiene 10% de FBS al tubo y se centrifuga el tejido a 2.500 rpm durante 2 min. </li><li> Resuspender el tejido en 2 ml de medio de co Neurobasalntaining 2% B27, y triturar el tejido de la médula espinal usando cada vez más pequeñas pipetas Pasteur pulidas al fuego. </li><li> Centrifugar las células a 2500 rpm durante 2 min, se resuspende en 2 ml de medio Neurobasal conteniendo B27, y las células de filtro con un filtro de células colador de 40 micras. </li></ol></li><li> Contar las células con un hemocitómetro y dividir las células por igual en dos tubos Eppendorf. </li><li> Centrifugar las células y eliminar completamente el sobrenadante (1-2 min tienen la obligación de retirar todo el sobrenadante con una punta baja de vacío) para los cócteles del factor de crecimiento no se diluirán permaneciendo sobrenadante después de la resuspensión celular. </li><li> Resuspender cada medio de las células en el fibrinógeno o la solución de trabajo de trombina que contiene factores de crecimiento, respectivamente, a una concentración de 250.000 células / l (recuentos sedimento celular durante aproximadamente ⅓ volumen final) y colocarlos en hielo antes del trasplante (Figura 1A). Tenga en cuenta que el fibrinógeno puede cuajar de forma espontánea cuando se mezclan con célulass antes de combinar con la trombina en el sitio de la lesión / injerto; para evitar la gelificación prematura, mezclar las células en fibrinógeno inmediatamente antes del injerto de células in vivo. </li></ol> 4. Trasplante <ol><li> Reexpose el lesionado T3 / 4 de la médula espinal dos semanas después de la transección de la médula espinal. </li><li> 5 l de fibrinógeno células y 5 trombina células mu l podrían ser inyectadas directamente en nueve puntos de la zona de la lesión a través del tejido de la cicatriz sellado y la duramadre intacta sin volver a abrir el sitio de la lesión. </li><li> Utilice una aguja 27 para crear 9 agujeros en la superficie del tejido de la cicatriz y la duramadre intacta 1-2 semanas después de la lesión: 3 en el centro (uno en el punto medio y dos en los laterales, y espaciados 0,5 mm entre sí) y 3 en tanto rostral y 3 en la interfaz de caudal (aproximadamente 0,5 mm rostral o caudal al centro de la lesión). </li><li> Se inyecta un medio volumen de solución de células fibrinógeno en tres puntos centrales de la zona de la lesión y un volumen de moneda en 3 puntos de la interfaz rostral y aqvolumen uarter en 3 puntos de la interfaz de caudal. </li><li> A continuación, inyecte inmediatamente células throbmbin en los mismos lugares. La mezcla de células de fibrinógeno y trombina células en el interior del sitio de la lesión se formará el gel de fibrina para mantener las células en lugar de la lesión y los cócteles de factores de crecimiento apoyará la supervivencia de las células. Las células se inyectaron utilizando pipetas de vidrio tirados conectados a un Picospritzer (General de válvula, Inc.). </li><li> Trasplante de NSC humanas cultivadas 21 en el mismo procedimiento descrito anteriormente, y el uso de fibrina y factor de crecimiento cócteles hechos a partir de reactivos humanos. </li></ol>

<ol>
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No tenemos nada que revelar.

Uno de los principales obstáculos para la NSC el trasplante de la médula espinal lesionada es pobre supervivencia en el centro de la lesión. Todos los huecos o cavidades en el sitio de la lesión potencialmente podrían reducir o disminuir la formación de relés neuronales funcionales entre axones supraespinales y segmentos de la médula espinal separados por debajo de la lesión. Además, la mala supervivencia de NSCs injertadas puede afectar a su integración con el tejido del huésped, y por lo tanto reducir la c...

Lesión de la médula espinal (SCI) a menudo daños no sólo tractos de sustancia blanca que llevan señales desde y hacia el cerebro, sino también la materia gris central, causando la pérdida segmentaria de interneuronas y las neuronas motoras. La consecuencia de la lesión medular es la pérdida de tanto la función motora y sensorial por debajo de la lesión. Desafortunadamente, el sistema nervioso central adulto (SNC) no espontáneamente a regenerar, dando como resultado déficits funcionales permanentes 1. Por lo tanto, la reconstrucción de la lesionada médula espinal adulta y la mejora de motor, sensorial y la función autonómica es un objetivo importante de la investigación de SCI. Las células madre neurales (NSC), ya sea directamente aislada de embrionarias o adultas del SNC, son células candidatos de peso para reemplazar las neuronas perdidas y glía. Por otra parte, estas células tienen el potencial para formar nuevos enlaces funcionales para restaurar la conducción axonal a través de un 2,3 sitio de la lesión. Hasta la fecha, no ha habido una elucidación detallada de la anatómica, electrophysiológica y efectos en el comportamiento de la formación de relé neuronal por NSCs trasplantados después severa SCI. Hay varias razones para esto: primero, NSCs trasplantados o tejido fetal CNS sobrevivir mal cuando se injertan en grandes cavidades de la lesión. Estudios previos muestran una pérdida sustancial de células temprano, dejando grandes cavidades lesión quística vacías 4,5. En algunos estudios, las células injertadas serían posteriormente se dividen y se llenan la cavidad de la lesión de 4,5, pero esto puede ocurrir después de un retraso de días o semanas, y posterior llenado sitio de la lesión podría no ser completa o coherente. En segundo lugar, un sistema de seguimiento eficaz que proporciona datos fiables sobre la supervivencia celular, la diferenciación / maduración, y el crecimiento de NSCs trasplantados era deficiente. La mayoría de los estudios iniciales utilizados anterógrada y retrógrada etiquetado para rastrear las proyecciones axonales de trasplantes de 2,3. Sin embargo, estas técnicas sólo parcialmente y, a menudo proyecciones axonales poco clara etiquetados derivados de células injertadas, y métodos de trazadores son subjetivost de artefactos causados ​​por fugas de tinte más allá de las células implantadas. Otros grupos utilizan marcadores neuronales específicos humanos para etiquetar las proyecciones axonales después del trasplante de NSC fetales humanos en roedores lesionada de la médula espinal 5,6. Sin embargo, en estos estudios, los xenoinjertos no sobrevivieron consistentemente bien. Recientemente, se utilizó la entrega viral del gen reportero GFP para etiquetar NSC cultivadas 7,8. Sin embargo, la expresión de GFP fue a menudo incoherente y puede ser regulado a la baja 7. Recientemente, el uso de ratones donantes transgénicas o ratas de forma estable que expresan el gen reportero, GFP, o la fosfatasa alcalina de placenta humana, ha mejorado espectacularmente el seguimiento de los trasplantados neurales células madre / progenitoras in vivo 9,11. En tercer lugar, varios estudios indican que in vitro NSCs rata cultivadas derivadas de cualquiera embrionarias o adultas del SNC se diferencian exclusivamente en linajes gliales cuando se trasplantan en el medio de la cor espinal adulta intacta o lesionadad 7,12,13, a pesar del hecho de que estas células madre neurales son capaces de diferenciarse en neuronas y glía tanto in vitro, lo que indica que los entornos locales pueden dictar el destino de las células madre. Alternativamente, NSC cultivadas, especialmente los derivados de SNC adulto, pueden tener propiedad intrínseca defaulty de diferenciarse en linajes gliales in vivo 13. Debido a las limitaciones mencionadas anteriormente, nuestro grupo ha desarrollado recientemente un nuevo protocolo para mejorar embrionario seguimiento NSC, la supervivencia y la diferenciación / maduración en el adulto con lesiones graves de la médula espinal. En pocas palabras, empezamos con una línea transgénica Fischer 344 ratas inbreed estable que expresa un gen reportero GFP que sustenta la expresión de GFP después in vivo del trasplante 14. A continuación, se utilizó NSC recién aisladas de día embrionario 14 Fischer 344 de la médula espinal, una etapa de desarrollo que conserva el potencial de generar ambas neuronas y glía. Finalmente, inmersosNSCs recién disociadas en una matriz de fibrina que contiene factores de crecimiento 15-17 para retener las células y distribuir uniformemente dentro de una gran cavidad de la lesión, con el objetivo de apoyar la supervivencia de las células del injerto, la diferenciación y la integración. Los injertos se colocan en sitios de T3 transección completa, dos semanas después de la lesión de la médula espinal. Estas células injertadas llenan constantemente sitios transección completa y diferenciarse en neuronas abundantes que se extendía un gran número de axones en la médula espinal de acogida a través de largas distancias 18. Se obtuvieron resultados similares usando cultivadas injertos de células madre neurales humanas a inmunodeficientes ratas 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

Las células madre neurales (NSC) pueden auto-renovarse y diferenciarse en neuronas y células gliales. NSCs trasplantados pueden reemplazar las neuronas perdidas y glía después de la lesión de la médula espinal (SCI), y pueden formar relés funcionales para volver a conectar los segmentos de la médula espinal por encima y por debajo de la lesión. Estudios previos de injerto de las células madre neurales han sido limitados por incompleta la supervivencia del injerto dentro de la cavidad de la lesión de la médula espinal. Además, el seguimiento de la supervivencia de las células del injerto, la diferenciación, y la extensión proceso no había sido optimizada. Finalmente, en estudios previos, se registraron típicamente NSC de rata en cultivo para diferenciarse en glía cuando injertado a la médula espinal lesionada, en lugar de las neuronas, a menos que el destino se debió a un tipo específico de célula. Para abordar estas cuestiones, hemos desarrollado nuevos métodos para mejorar la supervivencia, la integración y la diferenciación de NSC a sitios de incluso severa SCI. NSC estaban recién aisladas de embriones día 14 la médula espinal (E14) desde una línea transgénica de ratas Fischer 344 estable que expresa fl verdeproteína uorescente (GFP) y se incrusta dentro de una matriz de fibrina que contiene factores de crecimiento; esta formulación destinada a conservar las células injertadas en la cavidad de la lesión y apoyar la supervivencia celular. NSCs en el cóctel factor de fibrina / crecimiento se implantaron dos semanas después torácica nivel 3 (T3) completos transección de la médula espinal, lo que evita los períodos pico de la inflamación. Injertos resultantes llenan completamente la cavidad de la lesión y se diferenciaron en neuronas tanto, que se extendían los axones en el huésped de la médula espinal sobre notablemente largas distancias, y glía. Los injertos de NSCs humanos cultivados que expresan GFP produjeron resultados similares. Por lo tanto, los métodos se definen para la mejora de injerto neural de células madre, la supervivencia y el análisis de los resultados in vivo.

Etiquetado inmunohistoquímico de GFP demuestra que NSC rata injertado resuspendieron en tampón fosfato salino (PBS) (que carece de una matriz de fibrina y factores de crecimiento) sobrevivido mal en el sitio de la transección T3, sólo a la fijación de margen de la lesión / anfitrión y dejando la mayor parte del sitio de la lesión vacío sin células injertadas (Figura 2A). La supervivencia de NSC mejoró cuando se co-injertado con matrices de fibrina por sí solos, pero el injerto no llenar comp...

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

Matrices de fibrina que contienen factores de crecimiento se utilizaron para conservar las células madre neurales injertadas en los sitios de la transección completa de la médula espinal. Células injertadas llenaron completamente la cavidad de la lesión y diferenciarse en múltiples tipos de células neuronales, incluyendo las neuronas que se extendían axones en la médula espinal de acogida a través de largas distancias.

Promoción de la supervivencia y la diferenciación de células madre neurales con fibrina y factor de crecimiento cócteles después de la médula espinal severa lesión

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 vistas.  Veterans Administration Medical Center, San Diego.  Matrices de fibrina que contienen factores de crecimiento se utilizaron para conservar las células madre neurales injertadas en los sitios de la transección completa de la médula espinal. Células injertadas llenaron completamente la cavidad de la lesión y diferenciarse en múltiples tipos de células neuronales, incluyendo las neuronas que se extendían axones en la médula espinal de acogida a través de largas distancias. 

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.

La disección y la Cultura de las neuronas comisurales de médula espinal embrionaria

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

Investigación

Neurociencias

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.

Crecimiento y diferenciación de las células del hipocampo adulto ardilla de tierra del Ártico troncales nerviosas

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.

El trasplante quirúrgico de células madre neurales en la médula espinal de los ratones infectados con el virus neurotrópico Hepatitis Ratón

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.

Percusión fluido lateral: Modelo de Lesión Cerebral Traumática en ratones

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.

Lesiones de la Médula Espinal completo y Protocolo Disección del cerebro por Wholemount posterior<em&gt; In Situ</em&gt; La hibridación en larvas lamprea 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.

Una<em&gt; Ex Vivo</em&gt; Médula espinal inducida por láser Lesiones modelo para evaluar los mecanismos de degeneración axonal en tiempo 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.

Imágenes de la actividad neuronal en la corteza somatosensorial primaria usando Thy1-ratones transgénicos 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 ensayo desarrolló para evaluar la activación de células neuronales endógenos en un modelo murino de lesión 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.

Identificación de marcadores de superficie celular de células madre neuronales primarias y células progenitoras por etiquetado metabólico de sialoglicano

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.

Formación basada en una cinta rodante con la médula espinal lesionado ratas Wistar

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