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、異なるガス混合物への子犬12を公開します。チャンバは3-4仔マウスまたは1-2仔ラットを収容するために十分なサイズである4インチ径（容積= 450 ml）を、有しています。ここで、AIHプロトコルは、生後1-8（P1〜P8）歳の新生児に配信されます。 </li><li> 21％でチャンバーの酸素化を維持し、21％、「ベースライン」ガスを送達するために圧縮空気のガスタンクを使用してください。 「相対的な低酸素状態」を達成するために、10％酸素/ 90％窒素ガスのタンクを使用します。 NOTE：プラスチックチューブ（3/16 &quot;直径）は、流量計にガスタンクを接続しています。このように、流量計への入力は、ベースラインと低酸素気流のために一本のチューブそれぞれ構成されています。 </li><li>次のようにガス配管を構成します。 <ol><li>各チャンバーに1 L /分の流れを可能にするバイアス流調整器、流量計からのプラスチックチューブ（1/8 &quot;直径）を実行する（ すなわち 、4 L / minで4チャンバが使用される場合）。 </li><li>容積脈波室にバイアス流量調整からプラスチック管（直径1/8 &quot;）を実行します。各チャンバに1 L /分の空気流は、ガス交換は、生理学的および行動の観察に基づいて、動物に、非ストレス誘導されたものとみなさされる所定の流量です。 </li></ol></li><li>何のガス流が使用されていない、またはプロトコル中の脈波検査室のうち、他のチャンバに逃げるないようにキャップ付き室にバイアス流ユニットへとからすべての未使用の接続およびすべての未使用の開口部をシール。 </li><li> （室を置き、S）37℃に平衡化し、または体温を可能にするためにチャンバ内に動物を入れする前にインキュベーター。 </li></ol>断続的低酸素症のためのサイクルタイムの2キャリブレーション<ol><li> 、空気タンクと流量計との間で、すべてのチューブを検査し、適切な接続用計器とバイアス流ユニット、およびバイアス流ユニットとプレチスモグラフィーチャンバー（複数可）を流します。 例えば、使用されていない未使用のバイアス流ユニットのうちラインおよび任意のチャンバ開口部をチェック：すべての説明ラインと閉鎖の段階的チェックして、接続を確認し、手順1で説明したように接続が設定されています。 </li><li>手持ちO 2メートルで、周囲O 2センサを接続して、容積脈波室蓋にセンサーを取り付けます。 </li><li>両方のガスタンクを開き、ガスのオフサイクルの流れをクランプするプラスチック製のチューブで絶縁外科手術、長い取っ手止血のペアを1つ取り付けます。この方法では、一度に一つのガスタンク室あたり1リットル/分のガス流を供給しています。 「低酸素」ガス混合物をチャンバに供給される一方で、「ベースライン」のガス混合物をクランプ、およびその逆。 </li><li> 10％の目標レベルに到達し、21％に戻ることチャンバーO 2に必要な時間を記録します。その後のプロトコルの配信のための記録された時間を利用しています。 </li></ol>急性間欠性低酸素症の3管理<ol><li>ステップ2.1で説明したように、すべてのチューブとの接続を点検します。 </li><li>脈波検査室（複数可）に新生児を置き、チャンバーベースに容積脈波室の蓋を収容します。未使用室接続の適切なOリングシールにシールであるだけでなく、閉鎖を確認してください。これらの手順は、ガス混合物の適切な配送を確保するために重要です。 </li><li>子犬を保持室は37℃のインキュベーターに扱われることを置き、ドアを閉めます。室前のプロトコルを開始する37℃に平衡化することを許可します。 incubatで37℃で対照動物を維持または室内空気の酸素で。 </li><li>両方のガスタンクを開き、ガスのオフサイクルの流れをクランプするプラスチック製のチューブで絶縁外科手術、長い取っ手止血のペアを1つ使用します。この方法では、一度に一つのガスタンク室あたり1リットル/分のガス流を提供しています。 </li><li>正確に時間サイクルにハンドヘルドタイマーを使用し、したがって、チャンバへのガス流の交互に得られた、チューブ間の止血を切り替える時刻を示します。 </li><li> 2ステップ、20サイクルプロトコルのすべてのステップをオフにマークするために、図1に提供されるものなどの処理ログを使用して、各サイクルの完了を記録します。 </li><li>動物は指定範囲（37°Cになるインキュベーターの設定である33〜35°C）に維持されることを保証するために、サイクルごとの変更のインキュベーターの温度を監視します。 </li><li>密接にこのような発声、レベルの変化などの動物が見える苦痛なしでサイクルに耐えることを確認するために、プロトコル全体子犬の活動を監視四肢活性またはローリング。 </li></ol>脳室下帯から幹/前駆細胞の単離および培養4 注：コントロールに比べAIH次ニューロスフェア形成の変化は、組織や細胞レベルの変化を誘発するこのプロトコルの有効性を示すエンドポイントの例です。 <ol><li>セルを形成するニューロスフェアを単離するために、AIHが完了した直後にチャンバーからマウスまたはラット仔を削除します。プロトコル終端の30分以内にIACUCプロトコルに従って動物を生け贄に捧げます。 </li><li>以前の方法の章15,16と独立したビデオプロトコル8に発表されたように、SVZの組織を採取する神経球の文化に置き、標準の通路及びデリバティブニューロスフェアの展開を行っています。 </li></ol>

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The authors have nothing to disclose.

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><tbody><tr><td>Mouse plethysmography chambers</td><td>Buxco</td><td>PLY4211</td><td></td></tr><tr><td>Flow meter&amp;nbsp;</td><td>Porter</td><td>F150</td><td></td></tr><tr><td>Bias flow unit</td><td>AFPS</td><td></td><td></td></tr><tr><td>Baseline Gas Mix</td><td>Airgas</td><td>AIZ300</td><td>Compressed Air</td></tr><tr><td>Hypoxic Gas Mix</td><td>Airgas</td><td>X03NI72C2000189</td><td>10% Oxygen, balance nitrogen</td></tr><tr><td>Oxygen Meter</td><td>Teledyne</td><td>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

7.2K ビュー.  University of Florida. 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。 

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

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

生化神経建築研究における工学的神経前駆体の脳内移植

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

遺伝学

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

周産期虚血低酸素虚血性脳症モデル

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

生物学

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

新生児の脳室下帯のエレクトロ

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

神経科学

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

生体産後エレクトロポレーションおよびマウスの急性脳スライスにおける神経芽細胞移動のタイムラプスイメージング中

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

ラットにおける羊膜内リポ多糖で出生前低酸素性虚血を使用した未熟児のモデル化脳症

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

医学

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

期産期新生児ラットにおける幹細胞の気管内植在

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

新生児マウスにおける静脈注射

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

肺疾患を治療するための新生児げっ歯類における薬物の気管内点滴のための低侵襲方法

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

神経幹およびオリゴデンドロサイト前駆細胞単離のためのラット「脳搾乳」

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

著者スポットライト:Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

生きたラットからの神経幹細胞およびオリゴデンドロサイト前駆細胞の単離のための「脳搾乳」法

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

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生化神経建築研究における工学的神経前駆体の脳内移植

周産期虚血低酸素虚血性脳症モデル

新生児の脳室下帯のエレクトロ

生体産後エレクトロポレーションおよびマウスの急性脳スライスにおける神経芽細胞移動のタイムラプスイメージング中

ラットにおける羊膜内リポ多糖で出生前低酸素性虚血を使用した未熟児のモデル化脳症

期産期新生児ラットにおける幹細胞の気管内植在

新生児マウスにおける静脈注射

肺疾患を治療するための新生児げっ歯類における薬物の気管内点滴のための低侵襲方法

生きたラットからの神経幹細胞およびオリゴデンドロサイト前駆細胞の単離のための「脳搾乳」法

生きたラットからの神経幹細胞およびオリゴデンドロサイト前駆細胞の単離のための「脳搾乳」法