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).

注：このプロトコルのすべての動物の手順は、フロリダ大学の施設内動物管理使用委員会（IACUC）の承認を得て実施し、「実験動物の管理と使用に関する指針」に準拠しているされています。 1.基本実験は、断続的低酸素症の管理前に設定<ol><li>他の実験目的5,13,14のための大人の齧歯類とほぼ同じ方法で、全身マウスプレチスモグラフ室を使用して、異なるガ?...

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	<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...

この方法の目的は、再現性と効果的に新生児の齧歯類に全身の低下周囲の酸素の断続的な発作を提供することです。幹細胞生物学を操作するために、断続的低酸素症（IH）を使用するための理論的根拠は、増殖培地のO 2含有量が変更されたインビトロ細胞培養実験における由来します。具体的には、20％のO 2、3％O中の幹/前駆細胞集団の細胞増殖の増加は 2の結果の拡張された培養物の「標準的な」条件と比較した場合、アポトーシスの減少およびニューロン収量1,2を増加させました。 このグループは、全身IHの投与と豊富な経験を持っており、呼吸可塑3-7に IHの役割について鋭意検討しました。この作品、および慢性IHはげっ歯類CNS 8-10に神経新生を増加させることを最近の知見は、in vivoでの急性の探査のための基礎を形成プレコンディショニング刺激として低酸素状態（すなわち、組織の収穫に先立って）神経幹/前駆細胞（NPCの）11のその後の培養に。仔マウスは、急性間欠性低酸素症（AIH）の短い（&lt;1時間）の期間にさらされたときに驚くべき、脳室下帯（SVZ）から採取した細胞はかなりの神経球または接着単層細胞のような拡張のための容量を増加しました。 AIHプロトコルは、「ニューロンの運命」転写因子（Pax6の）の発現増加と関連していました。 従って、 インビボでの AIHプロトコルは、「プライム」のNPC培養前に手段を提供することができます。例えば、このアプローチのアプリケーションは、負傷した中枢神経系に移植前に細胞集団を拡大し、または単に以前のin vitro実験に培養細胞の神経分化を増加させる含めることができます。さらに、これは任意の、全身送達であるため、臓器、組織または細胞は、同様の研究のための候補です。したがって、記述されたようなプロトコルは、小型哺乳類で断続的な酸素操作に関する研究の広い範囲に適用できる可能性です。 このアプローチにはいくつかの利点があります。他の公開作品では、新生児を治療する前に、長期投与のために以下の取り扱いを可能にする低気圧チャンバ内のダム、とごみとして処理され、治療の9時の母体の接触を維持しました。現在のアプローチは、繁殖の女性、または各実験ごとに異なる​​ダムの使用に繰り返し治療をバイパスします。このプロトコルはまた、正確なゴミが一致したと年齢をマッチさせた新生児の研究を可能にします。代表的なデータは、このプロトコルの他のキーの強度、AIHとすなわち迅速性を実証し、配信として、神経幹細胞生物学における強力かつ一貫性のある生物学的応答を誘発します。これは、組織や細胞レベルのbiologiを引き出すために、このプロトコルのための先例を確立細胞生物学を変えるCAL変更。 このレポートではAIHにげっ歯類の子犬を露出するだけでなく、ニューロスフェアとして増殖SVZ細胞の集団の分析に使用する詳細な手順を概説します。

<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.

過去のデータに基づいて、初期の実験は、1分間のサイクル長を使用して行きました。上記のステップ2で実行される後続のキャリブレーションに基づいて、それは21％のベースラインに戻るには、同様の時間を要したこと、チャンバー内のO 2レベルは1分後に低酸素症のフラッシングで13％であったことを決定しました。しかし、2分間のサイクルを10％の酸素と、「ベースライン」サイ?...

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

この記事では、生後1-8マウスまたはラット仔に断続的低酸素症の短い期間を管理するための方法を説明します。このアプローチは、効果的に低酸素暴露の30分以内に収穫される培養神経前駆細胞の「影響をプライミング」強固な組織レベルを誘発します。

の配達<em&gt;インビボ</em&gt;急性間欠性低酸素症新生児げっ歯類で首相脳室下帯由来の神経前駆細胞の培養に

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

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

末梢血造血前駆細胞からヒト神経幹細胞の直接誘導

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

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

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

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不死化多能耳の前駆細胞に分化を開始

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

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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 ...

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一次繊毛を研究するために一次ニューロスフェア培養物を使用して

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

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血管内皮幹細胞の血管形成を視覚化するための新規乳腺脂肪移植技術

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

マウス胚における心臓前駆細胞集団の定量的ホール マウント免疫染色解析

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

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セルショウジョウバエ切り込みをトランスとの結合を評価する集計の試金-配位子とCisによるその阻害-配位子

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

大人の神経前駆細胞における P2X7 受容体を貪食、細孔の形成、カルシウム流入を調査するためのリアルタイム生細胞フローサイトメトリー

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

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