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).

LET OP: Alle dierlijke procedures in dit protocol worden uitgevoerd met toestemming van de Universiteit van Florida Institutional Animal Care en gebruik Comite (IACUC) en zijn in overeenstemming met de &quot;Gids voor de Zorg en gebruik van proefdieren. 1. Basic Experimentele ingesteld voordat intermitterende hypoxie Administration <ol><li> Expose pups 12 de verschillende gasmengsels met een hele lichaam muis plethysmograaf kamers op vrijwel dezelfde manier als volwassen knaagdi...

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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...

Het doel van deze methode is reproduceerbaar en effectief op intermitterende aanvallen van systemische verlaagd ambient zuurstof neonatale knaagdieren. De reden voor het gebruik van intermitterende hypoxie (IH) tot stamcelbiologie manipuleren is afkomstig van in vitro celkweek experimenten waarbij O 2 -gehalte van de groei media is veranderd. Specifiek in vergelijking met &quot;standaard&quot; condities van 20% O 2, verlengde kweek van stam / voorlopercellen cel populaties cellen in 3% O 2 leidt tot verhoogde proliferatie, apoptose verlaagde en verhoogde neuronale opbrengst 1,2. Deze groep heeft veel ervaring met de toediening van systemische IH, en heeft uitgebreide studies over de rol van IH uitgevoerd in de luchtwegen plasticiteit 3-7. Dit werk, en de recente bevinding dat chronische IH verhoogde neurogenese in het knaagdier CNS 10/08, vormt de basis voor de exploratie van acute in vivohypoxie als preconditionering stimulus (bijv. vóór weefsel oogsten) op de daaropvolgende kweek van neurale stam / voorlopercellen (NPC) 11. Opmerkelijk, wanneer de muis pups werden blootgesteld aan een korte (&lt;1 uur) periode van acute intermitterende hypoxie (AIH), cellen die uit de subventriculaire zone (SVZ) werden geoogst waren aanzienlijk verhoogde capaciteit voor expansie neurospheres of hechtende monolaag cellen. De AIH protocol werd ook geassocieerd met verhoogde expressie van een &quot;neuronale lot&quot; transcriptiefactor (Pax6). Dienovereenkomstig, in vivo AIH protocollen kan een middel tot &quot;prime&quot; NPC&#39;s voorafgaand aan de cultuur. Zo kan toepassingen hiervan zijn de groeiende celpopulaties vóór transplantatie in de gewonden centrale zenuwstelsel of simpelweg verhogen van de neuronale differentiatie van gekweekte cellen vóór in vitro experimenten. Verder, omdat een systemische afgifte, welkeorgaan, weefsel of cel een kandidaat voor soortgelijke studie. Daarom is het protocol zoals geschreven is potentieel van toepassing is op een breed scala van studies over intermitterende zuurstof manipulatie in kleine zoogdieren. Er zijn bepaalde voordelen aan deze aanpak. In andere gepubliceerde werk, werden pasgeborenen behandeld als een nestje met de dam in hypobaric kamers, die vóór het mogelijk maakt voor chronische toediening, minder handling op de behandeling, en onderhouden van de moeder contact tijdens de behandeling 9. De huidige benadering omzeilt herhaalde behandelingen voedingsbodem vrouwelijke, of het gebruik van een andere dam voor elk experiment. Dit protocol maakt het ook onderzoek naar precieze-nest op elkaar afgestemd en leeftijd gematchte pasgeborenen. Representatieve gegevens tonen een andere belangrijke kracht van dit protocol, namelijk de snelheid waarmee AIH, zoals geleverd, lokt een krachtige en consistente biologische respons in neurale stamcellen biologie. Dit wordt een precedent voor dit protocol om weefsel- en cellulaire niveau Biologisch wekkensche veranderingen die celbiologie veranderen. Dit verslag zal de gedetailleerde procedures voor het belichten knaagdieren pups AIH evenals de populatie analyse van SVZ cellen gekweekt als neurospheres schetsen.

<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.

De eerste experimenten, gebaseerd op historische gegevens, werden uitgevoerd met 1 min cycluslengten. Op basis van de volgende kalibreringen in stap 2 hierboven werd vastgesteld dat het O-niveau 2 in de kamer was 13% na 1 min na hypoxie spoelen en dat het duurde ongeveer gelijktijdig terugkeren naar de basislijn 21%. Echter, een 2 minuten cyclus was voldoende om zowel te bereiken 10% zuurstof en terugkeer naar 21% in de &quot;basislijn&quot; cyclus. Vervolgens werden 2 min cycluslengtes gebruikt. De duur prot...

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

Dit artikel beschrijft de methode voor het toedienen van een korte periode van intermitterende hypoxie tot postnatale dag 1-8 muis of rat pups. Deze aanpak effectief ontlokt een robuust weefsel niveau &quot;priming effect&quot; op gekweekte neurale stamcellen die binnen 30 minuten van de blootstelling hypoxie worden geoogst.

Levering van<em&gt; In Vivo</em&gt; Acute intermitterende hypoxie in Neonatale Knaagdieren Prime subventriculaire zone-afgeleide 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 ...

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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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