この作品は、米国心臓協会（AM）、米国国防総省（BC074986）、ボストン小児病院学部キャリア開発フェローシップ（TM、AM）からの資金によってサポートされていました。著者は、技術支援のためのアマンダ江とエリザベート江に感謝。

注：in vivoでの動物実験は、国立衛生研究所の実験動物の管理と使用に関する指針の推奨事項に厳密に従って実施した。プロトコルを見直し、ボストン小児病院の動物実験委員会によって承認された（プロトコル番号：13-10-2526R、14-02-2568R）。このプロトコルで使用されるすべての薬物は、医薬品グレードであり、これらの薬物は、無菌条件下で調製される。 1.フィブリンゲルの準備<ol><li> VEGFおよびbFGFが含まれているフィブリンゲルを準備します。 <ol><li>室温（25℃）に-80℃で保存されたフィブリノーゲンおよびトロンビ​​ンのストック溶液を解凍する。 </li><li>フィブリノゲンへのCaCl 2（最終濃度：45ミリモル）、VEGF（終濃度：0〜100 ng / ml）をおよびbFGF（0-100 / mlの最終濃度）のトロンビン（2.5 U / mlの最終濃度）を添加する溶液（最終濃度：0.9％sodiu 12.5 mg / mlの1.5mlチューブにおけるm食塩43-45）。 </li><li>ピペッティングにより穏やかに混合します。 </li><li>穏やかにp200とのピペットチップを用いて滴下様式で滅菌プラスチックシャーレ上に混合物200μlをピペット。 </li><li>彼らが固化するまで、30〜60分間、37℃で滴をインキュベートする。 注：固化ゲル注入（ 図1a）の前に数時間、室温（25℃）で密封されたプラスチックディッシュに保つことができる。 </li></ol></li><li>移植前に小さな外科用ハサミを用いて約3×3×3 mmの立方体にフィブリンゲルをトリミング。 </li></ol> 2.マウスの準備<ol><li>腹腔内ケタミンの（IP）注射（100mg / kg）およびキシラジン（10mg / kgの）によって成体マウス（8-12週）を麻酔し、マウスが十分にマウスのつま先をつまんで麻酔をかけていることを確認します。 <ol><li>実験中に乾燥を防ぐために、マウスの目に獣医軟膏を使用してください。 </li></ol></li><li>シェーブFUマウスの胸郭の左側の上R。 </li><li>マウスの気管内挿管を行います。 </li><li> 70°の角度をなす挿管スタンドにマウスを置き、スタンドの上部にある小さなゴムバンドの上に、その上部切歯をフックによって所定の位置にマウスを保持する。 </li><li>静かに鈍鉗子を用いて片側に舌を撤回。 </li><li>光ファイバグースネック顕微鏡照明装置を用いて喉頭を視覚化。 </li><li>気管内に気管内弾性カテーテル（21 G）を挿入します。 </li><li>マウスが自然発生的に（通常の100〜150呼吸/分、ない逆説的または浅い呼吸）がスムーズな方法で呼吸していることを確認してください。 </li><li>解剖顕微鏡下で発生しやすい位置にマウスを置きます。 </li><li>機械的にげっ歯類人工呼吸器（150呼吸/分、7ミリリットル/ kgの一回換気量）を使用して、マウスを換気する。 </li><li> 4 番目と5 番目の肋骨の間の肋間スペースを見つけるために肋骨を数える。 </li><li>エリアBの上に無菌のフィールドを作成します。yは徹底的にアルコールやポビドンヨードで拭く。無菌の外科用ドレープで適切に手術野をカバーしています。 </li></ol> 3.マウスの手術<ol><li>皮膚の0.25％ブピバカイン（200μl）を局所注射した後、解剖ハサミを使用して、肋間空間にわたって横断皮膚切開（約1cm長）を作る。 </li><li>肋間筋に0.25％ブピバカイン（200μL）を注入した後、細かい小さなハサミを使用して4 番目と5 番目の肋骨の間の筋肉の切開を作る。 </li><li>完全に左肺を可視化するために、リブの間の解剖リトラクターを挿入します。 <ol><li>細かい鉗子を使用して、左肺の中央の臓側胸膜の小さな領域（1×1mm角）をこすり。 </li><li>出血や空気の漏れが完全に制御されるまで滅菌綿棒を使用して領域に穏やかな圧力を適用します。 </li><li>フィブリノゲン/トロンビン（フィブリン糊）の新鮮な混合物（ステップ1.1.2。20＆＃少量を入れて181、p200とピペットチップを使用して領域にわたってリットル）。 </li></ol></li><li>優しくエリア（ 図1b）の上に小さな鉗子を用いて1フィブリンゲル（ステップ1.2）を配置。 <ol><li>ゲルが十分に肺の呼吸運動中にエリア上に固定されていることを確認します。 </li></ol></li><li>どちらも大規模な空気漏れや肺からの出血があることを確認してください。 </li><li>削除する必要はありません吸収性縫合糸、との緊密な切開（筋肉と皮膚層）。 </li><li>気胸を防ぐために27 G針および1mlシリンジを使用して胸腔を吸引する。 </li><li>機械的な換気を終了します。 </li></ol> 4.マウスの回復<ol><li>マウスが自然発生的にスムーズな方法（定期的な100〜150呼吸/分、ない逆説的または浅い呼吸）で呼吸していることを確認してください。 </li><li> IP脱水を防ぐために、予め温めておいた0.9％のNaClを1ml注入する。 </li><li>マウスは、循環温水パッド上で回復できるようにする。 </li><li> endotracheを削除マウスが安定した呼吸を持っていることを確認した後、他のチューブ。 </li><li>術後の鎮痛剤として3日間、メロキシカム（5 mg / kgを皮下注射（SC）を注入する。 </li><li>それは（その胃の上にロールバックすることが可能と直立のまま）胸骨で、意識的になるまで丁寧に15分間隔の最小のマウスの動きを監視します。 </li><li>回復後、手術をせずにマウスから単離された新しいケージにマウスを返す。 </li><li>毎日、以下の感染の兆候（発赤、腫脹、放電）、動物の基本的な生物学的機能（食品や水の摂取、排尿、排便、体重増加）だけでなく、臨床徴候苦痛のための手術部位（立毛、縮小運動）を監視外科的手技。 </li></ol> 5.収穫肺<ol><li> 7〜30日間、移植後、圧縮ガスの供給源を介してCO 2を使用して、マウスを安楽死させる。 </li><li>剣状突起の先端と胸骨の間に切開を作るノッチ（胸骨正中切開）と気管を切断し、心臓、肺、気管へのすべての接続を解剖することにより組織学的および生化学分析のために注入されたゲルと収穫肺全体。 </li><li> 、、4℃で一晩、4％パラホルムアルデヒド溶液で肺に移植されたゲルを固定OCT化合物に埋め込み、厚さ30μmの連続段部を取る。 </li><li>共焦点顕微鏡22,37,40を使用して（アクアポリン（AQP）5と界面活性タンパク質（SP）-B：CD31、上皮マーカー内皮マーカー）組織学的（ヘマトキシリン及びエオシン染色）および免疫組織化学的分析を実行します。 </li><li> 3D画像解析ソフトウェア37を使用して、肺の内皮および上皮細胞の三次元画像を形成する光学切片（厚さ30μm）のスタックをコンパイルする。 </li><li>画像解析ソフトウェア46を使用して、新しく形成された血管の投影面積を定量化する。 </li></ol>

The authors declare that they have no competing financial interests.

この記事では、成体マウスの生体の肺表面上の生体材料を注入するための新しい方法を紹介します。このシステムでは、ホスト肺由来の血管新生は、正常に材料内部に要約されている。このシステムは、研究者は内皮細胞、他の細胞（ 例えば 、上皮細胞、間葉系細胞、免疫細胞）とローカル血管新生50〜53と肺胞の再生24,54のために必要とされる様々なECM成分の間のク?...

このプロトコルの全体的な目標は、研究者は肺血管および肺発達の分子機構を特徴づけることができ、成体マウスの肺表面にフィブリンゲルを注入する方法を導入すること、及び可能な生体模倣材料を開発するためにこの知識を活用することである様々な肺疾患を治療するための生理的な肺の血管および肺胞形成を反復するの。 35以上の万人のアメリカ人が慢性閉塞性肺疾患および肺線維症を含む慢性肺疾患に苦しむ。これらの患者は大幅に日常生活1-3を損なうような息切れ、胸部圧迫感、しつこい咳、および疲労などの長期的な慢性呼吸器症状を持っている。これらの肺疾患のための効果的な治療法を開発する努力が多量にもかかわらず、現在、治療法は存在しない。そのため、これらの患者の生活の質が悪いと経済で、人的コストがhiであるGH 4-7。現在、肺移植は、末期慢性肺疾患を有する患者を保存する唯一の方法である。しかし、移植ドナー、高コスト、深刻な合併症、および低い生存率8-11の不足のため、移植が最適なアプローチではありません。組織工学技術の最近の急速な進歩は、前駆細胞または人工多能性幹（iPS）細胞12,13の様々なタイプの脱細胞化肺全体を再増殖することにより、移植可能な肺をbioengineerする研究者を可能にした。しかし、これらの生体工学肺が唯一の注入12,14,15後に数時間の宿主動物において機能的である。肺の複雑な構造および機能を再生するために生体材料を利用しても、かなり成功していない。成人の肺の再生を支配する主要な生物学的プロセスが十分に検討されていないためと考えられる。肺では、血管系の形成が早いと最も重要なイベントの一つ対応時間は限らですNGの開発と再生16-21。肺で新たに形成された血管系は、酸素、栄養素や器官形成に必要な各種の細胞成分を提供するだけでなく、細胞の22〜25の周囲に有益な調節シグナルを提供するだけでなく。このように、血管新生は、成人肺24,26,27回生肺胞形成において重要な役割を果たしている。また、調節解除された血管新生は、慢性閉塞性肺疾患（COPD）28、気管支肺異形成（BPD）、21-23、および肺線維症29のような慢性の肺疾患に寄与する。したがって、肺のエンジニアリングまたは慢性肺疾患を治療するためのより効率的な戦略を開発するためには、肺特異的血管形成の基本的メカニズムを理解する必要がある。 各器官は生理学的および病理学的条件30〜33の間で異なる場合がありますユニークな機械的および化学的特性を、表示されます。これらの器官特異microenvironメンツは、内皮細胞の挙動を調節し、器官特異的に24,34-36に血管網の形成を調整する。したがって、肺の再生のためのより効率的な戦略を開発するために、肺特異的血管形成の基礎となるメカニズムは、理解される必要がある。皮下ヒドロゲル移植のような従来のin vivoでの血管形成アッセイは、血管形成の研究37-39ために広く使用されてきたが、これらの方法は、器官特異的血管形成を再現しない。最近、マウス肺弾性モールドにマトリゲルを移植するための新規な方法が開発され、成功したゲル22中に血管および肺上皮細胞を動員することが示されている。このユニークなアプローチは、研究者は肺特異的血管形成の機構、ならびに生理的および病的状態における血管および非血管肺細胞間の相互作用を探索することを可能にする。 1）マトリゲルは臨床応用には適していないので。 2）Eゲルをキャストするために使用ラスチック金型は、ヒドロゲルおよびホスト肺組織との間の相互作用に影響を与える可能性があり、3）肺弾性モールドは、潜在的に、より臨床的に関連するアプローチ、血管新生因子を含む3次元フィブリンマトリックスとして、呼吸中に肺機能および疼痛障害を引き起こす（血管内皮増殖因子（VEGF）/塩基性線維芽細胞増殖因子（bFGF））、弾性モールドに鋳造することなく、マウス肺に移植された、成功したホスト肺由来の血管新生を要約されている。フィブリンゲル、トロンビン切断されたフィブリノゲンから生成されたポリマー原線維は、トラップする生体 40,41 で血管新生を促進させるようなbFGFおよびVEGFなどの血管新生因子の様々な知られている。 、その再生能力及び生分解性の性質42、フィブリンゲルが広く組織工学の分野で用いられている。 この記事では、生きているADULの肺の表面にフィブリンゲルを注入する新規かつユニークなアプローチを紹介しますトンマウスとホスト肺由来の血管新生は、in vivoでのゲルの内部に要約されていることを示している。肺特異的血管形成を研究するための研究を可能にし、この方法は、可能性肺疾患の様々なタイプの新しい治療アプローチの開発につながると有意に正常成体肺を再生する取り組みを進めます。

<table border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Name of Material/ Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fibrinogen from human placenta</td>
			<td>Sigma</td>
			<td>F4883</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T9549</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse VEGF 164</td>
			<td>R&#38;D</td>
			<td>493-MV</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse bFGF</td>
			<td>R&#38;D</td>
			<td>3139-FB</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Rodent Intubation Stand</td>
			<td style="width:177px;">Braintree Scientific INC</td>
			<td style="width:147px;">RIS 100</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fiber-Optic Light Source</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-565-35</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">20G Elastic catheter</td>
			<td>B.Braun</td>
			<td>4251652-02</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">MiniVent Ventilator</td>
			<td style="width:177px;">Harvard Apparatus</td>
			<td style="width:147px;">CGS-8009</td>
			<td>For ventilation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Stemi DV4 Steromicroscope</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-070-515</td>
			<td>For surgey</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Absobable suture</td>
			<td>Ethicon</td>
			<td>PDP304</td>
			<td>Surgical suture</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against CD31</td>
			<td>BD Biosciences</td>
			<td align="right">553370</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against AQP5</td>
			<td style="width:177px;">Abcam</td>
			<td>AB78486</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against SP-B</td>
			<td style="width:177px;">Millipore</td>
			<td>AB40876</td>
			<td>Immunohistochemistry</td>
		</tr>
	</tbody>
</table>

implantation of fibrin gel on mouse lung to study lung-specific angiogenesis

幹細胞研究と生物工学技術の最近の重要な進歩は、再生し、整形外科や歯周分野のシンプルな組織における損傷を修復するために生体材料を利用することで大きな進歩を遂げている。臓器再生の生物学的プロセスはよく検討されていないので、肺などのより複雑な三次元（3D）の構造と機能を再生しようとする臓器は非常に成功していない。これは、血管新生は、新しい血管の形成は、臓器再生において重要な役割を果たしていることが明らかになってきている。新たに形成された血管系は、酸素、栄養素や臓器再生のために必要とされる様々な細胞成分を提供するだけでなく、再生地元の組織に有益な信号を提供するだけでなく。首尾成人肺を再生成するため、それは血管新生が局所肺組織の再生を駆動する肺特異的微小環境を再現することが必要である。共同もののnventional インビボでのそのようなヒドロゲル（ 例えば 、フィブリンまたはコラーゲンゲルまたはマトリゲル-エンゲルブレス-ホルム-スウォームマウス肉腫細胞によって分泌ECMタンパク質の混合物）がリッチ細胞外マトリックス（ECM）の皮下移植などの血管新生アッセイ、広く探求するために利用される肺における生体材料の同所移植のための方法は十分に確立されていないため、血管新生の一般的なメカニズムは、肺特異的血管新生は、十分に特徴付けられていない。このプロトコルの目的は、ゲル内部ホスト肺由来の血管新生の成功要約を可能に、生きている成体マウスの肺の表面にフィブリンゲルを注入するユニークな方法を導入することである。このアプローチは、肺特異的微小環境が正常および病的両方の条件で血管新生および肺胞の再生を制御するメカニズムを探求する研究者を可能にします。注入された生体材料のリリース以来隣接lの物理的および化学的な信号を供給し、長官組織、罹患肺におけるこれらの生体材料の注入は、潜在的に肺疾患の様々なタイプの新しい治療アプローチを開発するための研究を可能にする、隣接した病変組織を正常化することができる。

ホスト肺由来の血管形成は、肺に移植生体材料の中に要約されているかどうかを調べるために、主要な血管新生因子のVEGFおよびbFGF（0、10および100ng / mlの各）を補充したフィブリンゲルのように生きたマウスの肺の表面に移植したマトリゲル22を用いて報告した。 図1aに示すように、これらの血管新生増殖因子を含むフィブリンゲル47を作製した。開胸後、左肺表...

Watch this Scientific Journal Video about Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis at JoVE.com

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

肺特異的血管形成を研究するためにマウスの肺にフィブリンゲルの移植

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

implantation-fibrin-gel-on-mouse-lung-to-study-lung-specific

Research

JoVE Journal

Medicine

10.0K ビュー.  Boston Children's Hospital and Harvard Medical School. Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse. 

ビデオ: Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

血管新生研究のための角膜ポケットアッセイのマウスモデル

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

医学

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

フィブリン塊における同種間葉系幹細胞の注入によってウサギの膝関節における骨軟骨欠損の治療

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

虚血ラットの肺の血管新生

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

マウス肺挿管の簡単な方法

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

マウス肺の病理生理学を研究するための非侵襲的気管内挿管

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

ヒアルロン酸ナトリウムの網膜下注入によるげっ歯類における網膜剥離モデル

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

挿管媒介気管内（IMIT）点眼：非侵襲的、肺特異的送達システム

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原発腫瘍と肺転移を研究するためのMEF細胞の単離

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

腫瘍の増殖、浸潤及び血管新生を研究するために、ニワトリでのヒト多発性骨髄腫異種移植モデルの確立

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring cardiomyocytes lost due to injury and disease. In contrast to the robust cardiac regeneration of certain lower vertebrates, adult mammalian hearts typically show minimal capacity for heart regeneration and repair. However, recent studies have sparked considerable scientific interest with the finding that, between postnatal day 1 to 7 (P1 to P7), the neonatal mouse heart retains significant regenerative potential after apical resection (i.e., surgical amputation and exposure of left ventricular apex). One major controversy over this finding might be due to the diverse surgery-related procedures used in efforts to replicate or expand upon this important finding. These instructions dynamically present the materials and methodology for apical resection in a mouse model. The salient steps of this rodent survival surgery involve hypothermia anesthesia, thoracotomy, surgical amputation of heart ventricular apex, and suture and recovery of mice. The approach described could expand the application of the apical resection mouse model for cardiovascular research.

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

アピカル切除マウスモデルは、初期の哺乳類の心臓の再生を研究するために

Cardiovascular disease plagues the whole world due to intensive lifestyle changes. Heart regeneration holds great promise for repairing and restoring ...