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).

OBS: Alla djurförsök i detta protokoll genomförs med godkännande av University of Florida Institutional Animal Care och användning kommittén (IACUC) och är i överensstämmelse med &quot;Guide för skötsel och användning av försöksdjur. 1. Grund Experimental Ställ in Innan Intermittent Hypoxi Administration <ol><li> Exponera valpar 12 till de olika gasblandningar med hjälp av en hela kroppen mus pletysmograf kammare på ungefär samma sätt som vuxna gnagare på and...

<ol>
	<li>Studer, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+proliferation,+survival,+and+dopaminergic+differentiation+of+CNS+precursors+in+lowered+oxygen.">Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.</a> J Neurosci. 20 (19), 7377-7383 (2000).</li><li>Chen, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+tension+regulates+survival+and+fate+of+mouse+central+nervous+system+precursors+at+multiple+levels.">Oxygen tension regulates survival and fate of mouse central nervous system precursors at multiple levels.</a> Stem Cells. 25 (9), 2291-2301 (2007).</li><li>Ling, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+intermittent+hypoxia+elicits+serotonin-dependent+plasticity+in+the+central+neural+control+of+breathing.">Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing.</a> J Neurosci. 21 (14), 5381-5388 (2001).</li><li>Mitchell, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invited+review:+Intermittent+hypoxia+and+respiratory+plasticity.">Invited review: Intermittent hypoxia and respiratory plasticity.</a> J Appl Physiol. 90 (6), 2466-2475 (2001).</li><li>Fuller, D. D., Zabka, A. G., Baker, T. L., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phrenic+long-term+facilitation+requires+5-HT+receptor+activation+during+but+not+following+episodic+hypoxia.">Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia.</a> J Appl Physiol. 90 (5), 2001-2006 (2001).</li><li>Fuller, D. D., Johnson, S. M., Olson, E. B., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+pathways+to+phrenic+motoneurons+are+enhanced+by+chronic+intermittent+hypoxia+after+cervical+spinal+cord+injury.">Synaptic pathways to phrenic motoneurons are enhanced by chronic intermittent hypoxia after cervical spinal cord injury.</a> J Neurosci. 23 (7), 2993-3000 (2003).</li><li>Baker-Herman, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+is+necessary+and+sufficient+for+spinal+respiratory+plasticity+following+intermittent+hypoxia.">BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia.</a> Nat Neurosci. 7 (1), 48-55 (2004).</li><li>Zhu, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+adult+rat+brain+after+intermittent+hypoxia.">Neurogenesis in the adult rat brain after intermittent hypoxia.</a> Brain Res. 1055, 1-6 (2005).</li><li>Zhang, J. X., Chen, X. Q., Du, J. Z., Chen, Q. M., Zhu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+exposure+to+intermittent+hypoxia+enhances+mice+performance+in+water+maze+and+8-arm+radial+maze+tasks.">Neonatal exposure to intermittent hypoxia enhances mice performance in water maze and 8-arm radial maze tasks.</a> Journal of neurobiology. 65 (1), 72-84 (2005).</li><li>Zhu, L. L., Wu, L. Y., Yew, D. T., Fan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hypoxia+on+the+proliferation+and+differentiation+of+NSCs.">Effects of hypoxia on the proliferation and differentiation of NSCs.</a> Mol Neurobiol. 31 (1-3), 231-242 (2005).</li><li>Ross, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+intermittent+hypoxia+elicits+enhanced+expansion+and+neuronal+differentiation+in+cultured+neural+progenitors.">In vivo intermittent hypoxia elicits enhanced expansion and neuronal differentiation in cultured neural progenitors.</a> Exp Neurol. 235 (1), 238-245 (2012).</li><li>Fuller, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+recovery+of+hypoxic+phrenic+responses+in+adult+rats+exposed+to+hyperoxia+for+the+first+month+of+life.">Induced recovery of hypoxic phrenic responses in adult rats exposed to hyperoxia for the first month of life.</a> J Physiol. 536 (Pt 3), 917-926 (2001).</li><li>Fuller, D. D., Fregosi, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatiguing+contractions+of+tongue+protrudor+and+retractor+muscles:+influence+of+systemic+hypoxia.">Fatiguing contractions of tongue protrudor and retractor muscles: influence of systemic hypoxia.</a> J Appl Physiol. 88 (6), 2123-2130 (2000).</li><li>Baker, T. L., Fuller, D. D., Zabka, A. G., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+plasticity:+differential+actions+of+continuous+and+episodic+hypoxia+and+hypercapnia.">Respiratory plasticity: differential actions of continuous and episodic hypoxia and hypercapnia.</a> Respir Physiol. 129 (1-2), 25-35 (2001).</li><li>Marshall, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+neurospheres+from+CNS+tissue.">Production of neurospheres from CNS tissue.</a> Methods Mol Biol. 438, 135-150 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> J Vis Exp. (45), (2010).</li></ol>

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 denna metod är att reproducerbart och effektivt leverera intermittent anfall av systemisk sänkt omgivnings syre till nyfödda gnagare. Den logiska grunden för att använda intermittent hypoxi (IH) att manipulera stamcellsbiologi kommer från i cellodlings vitro experiment där O2 halt av odlingsmediet ändras. Speciellt jämfört med &quot;vanliga&quot; villkoren för 20% O 2, förlängd kultur av stam / progenitorceller cellpopulationer celler i 3% O 2 resulterar i ökad spridning, minskad apoptos och ökad neuronal avkastning 1,2. Denna grupp har betydande erfarenhet av administration av system IH, och har genomfört omfattande studier om vilken roll IH i andnings plasticitet 3-7. Detta arbete, och den senaste upptäckten att kronisk IH ökad neurogenes i gnagare CNS 10/08, utgör grunden för utforskning av akut in vivohypoxi som konditionerings stimulus (dvs. före vävnads skörd) på den efterföljande kultur neurala stam / progenitorceller (NPC) 11. Anmärkningsvärt, när musen ungar utsattes för en kort (&lt;1 timme) period av akut intermittent hypoxi (AIH), celler som skördades från subventrikulära zonen (SVZ) hade signifikant ökad kapacitet för expansion som neurospheres eller vidhäftande monoskiktceller. Den AIH-protokollet var också förenad med ökad expression av en &quot;neuronal öde&quot; transkriptionsfaktor (Pax6). Följaktligen in vivo AIH protokoll kan tillhandahålla ett medel för att &quot;prime&quot; NPC före odling. Till exempel kan applikationer för detta tillvägagångssätt inkluderar expanderande cellpopulationer före transplantation in i skadade centrala nervsystemet, eller att helt enkelt öka neuronal differentiering av odlade celler före försök in vitro. Vidare, eftersom detta är en systemisk tillförsel, någonorgan, vävnad eller cell är en kandidat för liknande studie. Därför är det protokoll som skrivet potentiellt tillämplig på ett brett spektrum av studier på intermittent syre manipulation i små däggdjur. Det finns vissa fördelar med detta tillvägagångssätt. I andra publicerade arbeten, var nyfödda behandlas som en kull med dammen i hypobaric kammare, som gör det möjligt för kronisk dosering, mindre hantering före behandlingen, och underhållas moderns kontakt under behandlingen 9. Den nuvarande metoden kringgår upprepade behandlingar till en avelshona, eller användning av en annan damm för varje experiment. Detta protokoll kan också studera exakta kull-matchade och åldersmatchade nyfödda. Representativa data visar en annan viktig styrka detta protokoll, nämligen den snabbhet med vilken AIH, som levereras, framkallar en kraftfull och konsekvent biologiskt svar i neurala stamcellsbiologi. Det skapar ett prejudikat för detta protokoll för att framkalla vävnads- och cellnivå biological förändringar som förändrar cellbiologi. Denna rapport kommer att beskriva de detaljerade förfaranden som används för att exponera gnagare valpar till AIH samt populationsanalys av SVZ celler odlas som neurosfärer.

<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 initiala experiment, baserat på historiska data, genomfördes med hjälp av en min cykellängder. Baserat på de efterföljande kalibreringar som gjorts i steg 2 ovan bestämdes att O 2-nivå i kammaren var 13% vid 1 min efter hypoxi spolning och att det tog en liknande tid att återgå till baslinjen 21%. Det var en 2 min cykel är tillräcklig för att både uppnå 10% syre och en återgång till 21% under &quot;baseline&quot; cykel. Därefter har två min cykellängder använts. Protokollet varaktighet...

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

I den här artikeln beskrivs metoden för att administrera korta perioder av intermittent hypoxi till postnatal dag 1-8 mus eller råttungar. Detta tillvägagångssätt effektivt framkallar en robust vävnadsnivå &quot;priming effekt&quot; på odlade neurala stamceller som skördas inom 30 min av hypoxi exponering.

Leverans av<em&gt; In Vivo</em&gt; Akut intermittent hypoxi i neonatal Gnagare Prime subventrikulära zonen-härledda neurala stamceller kulturer

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

Research

JoVE Journal

Developmental Biology

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

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Direkt Induktion av humana neurala stamceller från perifert blod hematopoetiska progenitorceller

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Stamcellsliknande<em&gt; Xenopus</em&gt; Embryonala Explantat att studera tidig Neural Utvecklings funktioner<em&gt; In Vitro</em&gt; Och<em&gt; In Vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Initiera Differentiering i Immortaliserade multi Otic stamceller

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

Teknik till Target mikroinjektion till utvecklings<em&gt; Xenopus</em&gt; Njure

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Using Primary Neurosphere Cultures to Study Primary Cilia

Använda Primära neurosfärkulturer att studera Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

En ny mammografisk feta padtransplantationsteknik för att visualisera fartygsgenerering av vaskulära endotelceller

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

Kvantitativa hela-mount immunofluorescens analys av hjärt stamceller populationer i musembryon

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation analyser att utvärdera bindningen av Drosophila skåran med Trans-ligander och dess hämning av Cis-ligander

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Realtid Live-cell flödescytometri att undersöka kalcium tillströmning, Pore bildandet och fagocytos av P2X7-receptorer i vuxen neurala stamceller

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Kontrollerad kortikal effekt modell av mus hjärnskada med terapeutisk transplantation av humana inducerade pluripotenta stamceller-härledda neurala celler

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...