Name: delivery of in vivo acute intermittent hypoxia in neonatal rodents to prime subventricular zone-derived neural progenitor cell cultures
Uploaded: 2015-11-02T15:00:00
Description: Watch this Scientific Journal Video about Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures at JoVE.com

The authors acknowledge funding sources responsible for this work: 5K12HD055929 (HHR), 5R01NS080180-02 (DDF).

NOTA: Todos los animales procedimientos en este protocolo se llevan a cabo con la aprobación de la Universidad de Florida Institucional Cuidado de Animales y el empleo Comisión (IACUC) y están en cumplimiento de la &#39;Guía para el Cuidado y Uso de Animales de Laboratorio &quot;. 1. Experimental básico establecido antes de la administración intermitente Hipoxia <ol><li> Expose cachorros 12 a las diferentes mezclas de gas usando un conjunto de cuerpo cámaras de pletismóg...

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This work reports the development of a protocol to expose neonatal rodents to AIH. The parameters described here effectively alter in situ neural stem cell biology, which is observable over several rounds of cell passage. Specifically, AIH increases the number of non-adherent neurospheres, the expansion of cells within each neurosphere (refected by sphere diameter), the expansion of adherent NPC populations, and the presence of neuroblasts in both non-adherent and adherent populations. It should be emph...

El objetivo de este método es para entregar de forma reproducible y eficaz episodios intermitentes de oxígeno ambiental sistémica rebajado a los roedores neonatales. La justificación del uso de la hipoxia intermitente (IH) para manipular la biología de células madre se origina a partir de experimentos de cultivo celular in vitro en el que se altera O 2 contenido del medio de crecimiento. En concreto, si se compara con las condiciones &quot;estándar&quot; de 20% de O 2, cultivo prolongado de células madre / progenitoras células poblaciones celulares en 3% O 2 resultados en un aumento de la proliferación, la disminución de la apoptosis y el aumento de 1,2 rendimiento neuronal. Este grupo tiene una gran experiencia con la administración de IH sistémica, y ha realizado extensos estudios sobre el papel de IH en la plasticidad respiratoria 3-7. Este trabajo, y el hallazgo reciente que IH crónica aumenta la neurogénesis en el roedor CNS 8-10, constituye la base para la exploración de aguda in vivohipoxia como estímulo acondicionamiento previo (es decir., antes de la cosecha de tejidos) en el posterior cultivo de células madre / progenitoras neuronales (CPN) 11. Sorprendentemente, cuando crías de ratón fueron expuestos a un breve período (&lt;1 hr) de la hipoxia intermitente aguda (AIH), las células que fueron cosechadas de la zona subventricular (SVZ) habían aumentado significativamente la capacidad para la expansión como neuroesferas o monocapa de células adherentes. El protocolo AIH también se asoció con un aumento de la expresión de un factor de &quot;destino neuronal&quot; transcripción (Pax6). En consecuencia, en vivo protocolos AIH pueden proporcionar un medio para &quot;prime&quot; NPCs antes del cultivo. Por ejemplo, las solicitudes de este enfoque podrían incluir la expansión de poblaciones de células antes del trasplante en el sistema nervioso central lesionado, o el simple aumento de la diferenciación neuronal de células cultivadas antes de los experimentos in vitro. Además, dado que se trata de una administración sistémica, cualquierórgano, tejido o célula es un candidato para el estudio similar. Por lo tanto, el protocolo como escrito es potencialmente aplicable a una amplia gama de estudios sobre la manipulación de oxígeno intermitente en pequeños mamíferos. Hay ciertas ventajas de este enfoque. En otro trabajo publicado, los neonatos fueron tratados como una camada con la presa en cámaras hipobárica, que permite la dosificación crónica, menos manipulación antes del tratamiento, y se mantienen contactos materna durante el tratamiento 9. El enfoque actual no pasa por tratamientos repetidos a una hembra reproductora, o el uso de una presa diferente para cada experimento. Este protocolo también permite el estudio de los recién nacidos de basura de concordancia y de la misma edad precisas. Los datos representativos demuestran otra fortaleza clave de este protocolo, es decir, la rapidez con la que AIH, como entregado, provoca una respuesta biológica potente y consistente en la biología de las células madre neurales. Esto establece un precedente para este protocolo para obtener biologi tejidos y nivel celularCal cambios que alteran la biología celular. Este informe se esbozarán los procedimientos detallados utilizados para la exposición de las crías de roedores a AIH, así como el análisis de la población de las células SVZ crecido como neuroesferas.

<table border="0" cellpadding="0" cellspacing="0" width="768">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:243px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Mouse plethysmography chambers</td>
			<td style="width:147px;">Buxco</td>
			<td style="width:136px;">PLY4211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Flow meter&#160;</td>
			<td style="width:147px;">Porter</td>
			<td style="width:136px;">F150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Bias flow unit</td>
			<td style="width:147px;">AFPS</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Baseline Gas Mix</td>
			<td style="width:147px;">Airgas</td>
			<td style="width:136px;">AIZ300</td>
			<td>Compressed Air</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hypoxic Gas Mix</td>
			<td style="width:147px;">Airgas</td>
			<td style="width:136px;">X03NI72C2000189</td>
			<td>10% Oxygen, balance nitrogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Oxygen Meter</td>
			<td style="width:147px;">Teledyne</td>
			<td style="width:136px;">AX-300</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of in vivo acute intermittent hypoxia in neonatal rodents to prime subventricular zone-derived neural progenitor cell cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Los experimentos iniciales, basados ​​en datos históricos, se realizaron utilizando longitudes de ciclo 1 min. Sobre la base de las calibraciones posteriores realizadas en el Paso 2 anterior, se determinó que el nivel de O 2 en la cámara era 13% en 1 min de lavado post-hipoxia y, que tomó un tiempo similar para volver a la línea de base 21%. Sin embargo, un ciclo de 2 min era suficiente para lograr tanto 10% de oxígeno y un retorno a 21% durante el ciclo de &quot;línea base&quot;. Posteriormente, s...

Watch this Scientific Journal Video about Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures at JoVE.com

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

En este artículo se describe la metodología para la administración de cortos períodos de hipoxia intermitente para postnatales días 1-8 de ratón o rata crías. Este enfoque suscita efectivamente un nivel de tejido robusto &quot;efecto priming&quot; en células progenitoras neurales cultivadas que se cosechan en los 30 minutos de exposición a la hipoxia.

Entrega de<em&gt; En Vivo</em&gt; Aguda intermitente hipoxia neonatal en roedores a derivados de la Zona Prime subventricular progenitoras neurales Cultivos Celulares

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

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Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

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