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).

BEMÆRK: Alle dyreforsøg i denne protokol udføres med godkendelse fra University of Florida Institutional Animal Care og brug Udvalg (IACUC), og er i overensstemmelse med den »Guide til Pleje og anvendelse af forsøgsdyr. 1. Grundlæggende Eksperimentel oprettet inden Intermitterende Hypoxi Administration <ol><li> Expose unger 12 til de forskellige gas blandinger ved hjælp af en hel-krop mus plethysmograph kamre på samme måde som voksne gnavere for andre eksperimentelle fo...

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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...

Målet med denne metode er at reproducerbart og effektivt levere intermitterende anfald af systemisk sænket omgivende ilt til neonatale gnavere. Begrundelsen for at bruge intermitterende hypoxi (IH) til at manipulere stamcellebiologi stammer fra in vitro-cellekultur forsøg, hvor O 2 indhold vækstmediet ændres. Konkret i forhold til &quot;standard&quot; betingelser for 20% O 2, udvidet kultur af stamceller / stamceller populationer celler i 3% O 2 resultater i øget spredning, nedsat apoptose og øget neuronal udbytte 1,2. Denne gruppe har betydelig erfaring med administration af systemiske IH, og har gennemført omfattende undersøgelser om betydningen af IH i respiratorisk plasticitet 3-7. Dette arbejde, og den seneste konstatering af, at kronisk IH øget neurogenese i gnaver CNS 8-10, danner grundlag for udforskning af akut in vivohypoxi som forkonditionering stimulus (dvs.., før vævet høst) af den efterfølgende kultur af neurale stamceller / progenitorceller (NPCs) 11. Bemærkelsesværdigt, når musen unger blev udsat for en kort (&lt;1 time) periode med akut intermitterende hypoxi (AIH), celler, der blev høstet fra den subventrikulære zone (SVZ) havde signifikant øget kapacitet til udvidelse som neurosfærer eller omliggende monolag celler. Den AIH protokol blev ligeledes forbundet med forøget ekspression af et &quot;neuronalt&quot; transkriptionsfaktor (Pax6). Følgelig in vivo AIH protokoller kan tilvejebringe et middel til at &quot;prime&quot; NPC før dyrkning. For eksempel kunne applikationer til denne fremgangsmåde, omfatter ekspanderende cellepopulationer før transplantation ind i såret centralnervesystemet eller blot at øge neuronal differentiering af dyrkede celler før in vitro-forsøg. Endvidere fordi dette er en systemisk levering, enhverorgel, væv eller celle er en kandidat til lignende undersøgelse. Derfor protokollen som skrevet er potentielt anvendelig til en bred vifte af undersøgelser af intermitterende ilt manipulation i små pattedyr. Der er visse fordele ved denne fremgangsmåde. I andre offentliggjorte arbejde blev nyfødte behandlet som et kuld med dæmningen i lavtryksforhold kamre, som giver mulighed for kronisk dosering, mindre håndtering før behandling, og vedligeholdt mødres kontakt under behandling 9. Den nuværende fremgangsmåde omgår gentagne behandlinger til avlshun, eller anvendelsen af ​​en anden dæmning for hvert forsøg. Denne protokol tillader også undersøgelse af præcise kuld-matchede og aldersmatchede nyfødte. Repræsentative data viser et andet centralt styrken af ​​denne protokol, nemlig den hurtighed, hvormed AIH, som leveret, fremkalder en stærk og sammenhængende biologisk respons i neural stamcelle biologi. Dette etablerer en præcedens for denne protokol til at fremkalde vævs- og cellulært niveau biological ændringer, der ændrer cellebiologi. Denne rapport vil skitsere de detaljerede procedurer, der anvendes til at udsætte gnaver hvalpe til AIH samt befolkningen analyse af SVZ celler dyrkes som neurosfærer.

<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 indledende forsøg, baseret på historiske data, blev udført ved hjælp af 1 min cyklus længder. Baseret på de efterfølgende kalibreringer udført i trin 2 ovenfor, blev det bestemt, at O 2-niveau i kammeret var 13% ved 1 min post-hypoksi skylning og, at det tog en lignende tid til at vende tilbage til 21% baseline. Men en 2 min cyklus var tilstrækkelig til både at opnå 10% ilt og en tilbagevenden til 21% i løbet af &quot;baseline&quot; cyklus. Efterfølgende er 2 min cykluslængder blevet anvendt....

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

Denne artikel beskriver den metode til administration korte perioder af intermitterende hypoxi til postnatal dag 1-8 mus eller rotteunger. Denne tilgang effektivt fremkalder en robust vævsniveau &quot;priming effekt&quot; på dyrkede neurale stamceller, der er høstet inden for 30 minutter af hypoxi eksponering.

Levering af<em&gt; In vivo</em&gt; Akut intermitterende Hypoxi i Neonatal Gnavere til premierminister subventrikulære Zone-afledte Neural progenitorcelle kulturer

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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