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).

MERK: Alle dyr prosedyrer i denne protokollen er utført med samtykke fra University of Florida Institutional Animal Care og bruk Committee (IACUC) og er i samsvar med &quot;Guide for omsorg og bruk av forsøksdyr&quot;. 1. Grunnleggende Experimental satt opp før Intermittent Hypoksi Administration <ol><li> Expose valper 12 til de forskjellige gassblandinger ved hjelp av en hel-kropp mus plethysmograph kamre mye på samme måte som voksne gnagere for andre eksperimentelle form?...

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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 metoden er å reproduserbart og effektivt levere periodiske utbrudd av systemisk senket ambient oksygen til nyfødte gnagere. Begrunnelsen for bruk av intermitterende hypoksi (IH) for å manipulere stammen cellebiologi stammer fra in vitro cellekultur eksperimenter hvor O 2 innholdet i vekstmediet blir endret. Nærmere bestemt, når sammenlignet med &quot;standard&quot; betingelser 20% O 2, utvidet kultur av stammen / progenitor-cellepopulasjoner celler i 3% O 2 resulterer i økt proliferasjon, apoptose redusert og øket neuronal utbytte 1,2. Denne gruppen har betydelig erfaring med administrasjon av systemisk IH, og har gjennomført omfattende studier på rollen IH i luft plastisitet 3-7. Dette arbeidet, og den nylige funn at kronisk IH økt neurogenesis i gnager CNS 8-10, danner grunnlag for utforskning av akutt in vivohypoksi som en preconditioning stimulus (f.eks., før vev høst) på den påfølgende kultur av neural stilk / stamceller (NPC) 11. Bemerkelsesverdig, når museunger ble utsatt for en kort (&lt;1 time) periode av akutt intermittent hypoksi (AIH), celler som ble høstet fra subventricular sonen (SVZ) hadde betydelig økt kapasitet for ekspansjon som neurosfærer eller adherente monolagsceller. Den AIH protokollen ble også forbundet med økt ekspresjon av en &quot;neuronal edge&quot; transkripsjonsfaktor (Pax6). Følgelig in vivo AIH protokoller kan være et middel for å &quot;prime&quot; NPCer før kultur. For eksempel kan programmer for denne fremgangsmåten omfatter å utvide cellepopulasjoner før transplantasjon inn i det skadede sentralnervesystemet, eller ganske enkelt å øke neuronal differensiering av dyrkede celler før in vitro eksperimenter. Videre, fordi dette er en systemisk tilførsel, noeorgan, vev eller celle er en kandidat for lignende studie. Derfor er protokollen som er skrevet potensielt gjelder for et bredt spekter av studier på intermitterende oksygen manipulasjon i små pattedyr. Det er visse fordeler med denne tilnærmingen. I andre publiserte arbeider, ble nyfødte behandlet som et kull med demningen i hypobar kamre, som gjør det mulig for kronisk dosering, mindre håndtering før behandling, og opprettholdt morens kontakt under behandling 9. Den aktuelle tilnærmingen omgår gjentatte behandlinger til en kvinnelig avl, eller bruk av en annen demning for hvert eksperiment. Denne protokollen tillater også studier av presise søppel-matchet og alderstilpassede nyfødte. Representative data viser en annen viktig styrke denne protokollen, nemlig hurtighet som AIH, som levert, utløser en kraftig og konsekvent biologisk respons i nevrale stamcellebiologi. Dette etablerer en presedens for denne protokollen for å lokke fram vevs- og cellenivå biological endringer som endrer cellebiologi. Denne rapporten vil skissere de detaljerte prosedyrer som brukes for å utsette gnager valpene til AIH samt befolkningen analyse av SVZ celler dyrket 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 første forsøkene, basert på historiske data, ble utført ved bruk av en min syklus lengder. Basert på de påfølgende kalibreringer utført i trinn 2 ovenfor, ble det fastslått at O 2 nivået i kammeret var 13% ved 1 min post-hypoksi spyling, og at det tok en lignende tid til å vende tilbake til 21% baseline. Men en 2 min syklus var nok til å både oppnå 10% oksygen og en tilbakevending til 21% i løpet av &quot;baseline&quot; syklus. Senere har 2 min sykluslengder blitt brukt. Protokollen varighet...

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 artikkelen beskriver metodikk for å administrere korte perioder av intermittent hypoksi til postnatal dag 1-8 mus eller rotteunger. Denne tilnærmingen effektivt utløser en robust vev nivå &quot;priming effekt&quot; på dyrkede nevrale stamceller som er høstet i løpet av 30 minutter av hypoksi eksponering.

Levering av<em&gt; I Vivo</em&gt; Akutt Intermittent Hypoksi i Neonatal Gnagere til Prime subventricular Zone-avledet Neural stamcelle 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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Developmental Biology

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

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