著者らは、肺血流のテクニックで彼の忠告博士Luborベルジッヒ（生理学研究所、チューリッヒ大学）に感謝したいと思います。この作品は、カントンチューリッヒのKrebsliga、ウォルターL.とヨハンナウルフ財団、チューリッヒ、リディアHochstrasser財団、チューリッヒ、スイス国立科学財団、SNF、スイス、Schweizerischer法令に基づく連合組織体Balgrist、大学からの補助金によって支えられてチューリッヒの。

1 のIn-situ肺血および固定<ol><li> 112.5 mg / kgの（体重）ケタミン、16.5 mg / kgのキシラジン、および15 mg / kgのアセプロマジン（または適切な鎮痛剤を含む他の麻酔による）を含む緩衝化生理食塩水（PBS）〜150μlのリン酸塩の腹腔内注射によりマウスを麻酔。 </li><li>マウスの反射神経は、もはや観察されていない場合、バックダウン手術台の上にそれを修正し、なでつける毛に70％エタノールで毛皮を濡らす。 </li><li>首に腹部から皮膚を開き、両側に引っ張ったり、それを削除します。 </li><li>腹膜と十分な大きさに胸を開きます。その後の灌流の効率低下を避けるために大きな血管（静脈の脈など ）の破裂を防ぐ。 </li><li>肝臓の下に下大静脈尾側をカットします。 </li><li>肺は完全に白であるとまでは鼓動する心臓の右心室にゆっくりと10〜15ミリリットルのPBSを注入するために24G針 - 21Gを搭載した20ミリリットルの注射器を使用し心臓が（心静止）暴行を停止します。 </li><li>血管クランプで肝臓と横隔膜上の血管をピンチオフ。 </li><li>右心室にPBS中の3％パラホルムアルデヒド〜2ミリリットル（PFA）を注入する注射針24G - 21Gを搭載した10 mlの注射器を使用しています。 </li><li>胸郭の外気管を発見。肺が膨張するまで気管に頭蓋のPBS中の3％PFA（胸郭の外側）の約3ミリリットルを注入するために、同じ注射器を使用しています。直ちに針を除去した後、血管クランプで穿刺の気管尾をピンチオフと固定を可能にするために10分間待ちます。 </li><li>血管クランプ上記血管を横断し、クランプを取り外し、過剰PFAを削除するには右心室に〜2mlのPBSを注入します。 </li><li>穿刺針を持つすべての肺葉の下の部分は、気管に血管クランプを取り外して、中のPFAまで初回穿刺の気管尾に培地/ PBSで1:1を埋め込むPolyFreezeのソリューションの3〜5 mlを注入する肺ローブは、ソリューションに置き換えられます。 </li><li>鉗子で心を引いて、結合組織の靭帯、血管や気管をtransectingによって注意深く肺を取り外します。肺に接続されている心を保つ。 </li><li>肺組織分析の進め方に応じてセクション2または3に概説プロトコルに進みます。あなたは両方の分析を行いたい場合は、1つの肺葉を削除して、3で説明したように進みます。 2で説明したように心臓に接続、残りの葉を処理します。 </li></ol> 2。全体肺葉における lacZ-タグ転移性腫瘍細胞の可視化<ol><li>タイトな開閉蓋のプラスチックカップに肺を置き、30〜60分間、室温でPBS中2％ホルムアルデヒドで固定します。 </li><li>固定液を取り除き、PBSで十分に3回洗浄します。 </li><li>新たに5 - ブロモ-4 - クロロ-3 - インドリル-β-D-ガラクトシド（X-Gal）を染色液（X-galストック溶液[40 mg / mlで製造された少なくとも10ミリリットルを追加ジメチルホルムアミド] 1時40分基本的な染色液で希釈し、pHは7.1（ 表1）、光から保護する）。 </li><li>溶液中で完全にそれを維持し、空気の交換を可能にするために鍋でのみゆるく蓋を置くスイミング肺の上にガーゼを入れてください。 </li><li>光から保護して3から5時間37℃でインキュベートする。 </li><li> X-gal溶液を取り除き、残留X-gal溶液（オプション）を削除し、4％PFAを追加するには、PBSで一回すすいでください。 </li></ol> 3。肺凍結切片における lacZ-タグ転移性腫瘍細胞の可視化<ol><li>原液PolyFreeze埋め込み培地で1月3日にプレフィルラベルの付いた埋め込み型。上に肺葉を入れて、葉が完全に覆われるまで、メディアを埋め込んでいっぱい。気泡がないように配慮してください。 </li><li> 20分間、4℃で埋め込まれた肺をインキュベートする。 </li><li>その後-80℃でゆっくりとドライアイスとイソペンタンと店舗の混合物中に埋め込ま肺を凍結</li><li> 7から10μmの凍結をカットクライオスタットのセクションとSuperFrostPlus顕微鏡スライド上にマウントします。 </li><li>加湿チャンバー内で24時間37℃でのX-Gal染色液で直ちにセクションをインキュベートする。 </li><li>蒸留水で簡単にその後5分間のために2回、PBSでスライドを洗浄します。 </li><li> 10から30秒のための核ファーストレッドで対比染色、蒸留水ですすぎ、免疫マウントにスライドをマウントします。 </li></ol> 4。代表的な結果浅井ら高転移LM8サブ細胞株由来のものとは異なる親のダン細胞由来の皮下原発腫瘍が、自発的に検出可能な肺転移を形成しないことをダン由来LM8 OSのマウスモデルの元の記事で報告同系のC3Hマウス14。ここで報告技術により、我々はDunn/LM8マウスのOSモデルにおける転移の形成を再調査。我々は安定したlacZの形質ダンとLM8細胞を利用しましたsおよびマウスでのin-situ肺血流および固定のためのプロトコル。 lacZを形質導入し、非形質制御ダンとLM8細胞は図1に示されている皮下注射したマウスの灌流および非灌流肺の代表画像。制御ダン細胞を注射したマウスでは、肉眼的および顕微鏡的転移は非灌流灌流した肺（ 図1A、I-IV）で検出不可能であった。しかし、興味深いことに、ダン-lacZの細胞を注射したマウスでは、X-gal染色は非灌流肺（ 図1A、vi）の表面上の単一細胞や小さな細胞クラスター（&lt;0.1 mm）の青微小転移病巣を明らかにした。 イン肺の in situ灌流および固定がさらにダン-lacZを微小転移（ 図1A、ⅷ）の検出を改善しました。しかし、巨視的巣に伸長（ 図1A、V、VII）は観察されなかった。&lt;/ P&gt; 直径0.1mm以上LM8細胞、半透明の、かろうじて検出macrometastatic巣大きなコントロールを注射したマウスでは、非肺灌流した（ 図1B、i）で認識された。肺の灌流（ 図1B、iii） は病巣の検出を改善しなかった。しかし、マウスで複数のLM8-lacZの細胞を注入しX-Galを青く染色マクロ（ 図1B、v）と微小転移（ 図1Bは、vi）が非灌流した臓器の表面に検出された。また、肺の血流はさらにマクロおよび微小転移（ - VIII 図1B、vii）の検出能を向上させました。その結果、マイクロおよびmacrometastasesは主として臓器表面の下に巣も見えるようになっている組織灌流さの半透明に、より高い密度とより多くの目に見えるようになった。 追加のH肺組織の凍結切片を用いた分析は 、lacZ istological形質ダンとLM8細胞を注射したマウスの肺転移の検出能の改善を確認した。ダン-lacZまたはLM8-lacZを細胞に由来する原発腫瘍を持つマウスでは、それぞれのコントロール細胞、微小転移あるいは単一細胞病巣の原発腫瘍を持つマウスと異なり肺切片（ 図2）に認識された。また、macrometastasesも制御LM8細胞（ 図2C、D）を注射した動物に比べLM8-lacZを細胞を注射したマウスで、よりはっきりと見えた。 <img alt="図1" src="/files/ftp_upload/4162/4162fig1.jpg" /> 図 （I、II、V、VI）が非灌流灌流し、非形質導入（I〜IV） および lacZ形質導入（V-VIII）ダン（）とLM8（B）細胞の自発的な転移の1。検出C3Hマウスにおいて（III、IV、VII、VIII）肺。代表のX-Gal-STAへの転移ined全体の肺（v、およびBのvii）及びそれぞれのクローズアップ（VI、VIIIでAおよびB）を青色で表示されます。非タグ付けLM8細胞（BにおけるI-IV）を注射したマウスの臓器内Macrometastatic病巣が（&gt; 0.1ミリメートル）は、アスタリスクによって示されます。クローズアップに示す領域が全体の肺のための代表的なものである。スケールバーは全体の肺の画像で1ミリメートルとクローズアップさ0.1mmを示す。 <a href="http://www.jove.com/files/ftp_upload/4162/4162fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img alt="図2" src="/files/ftp_upload/4162/4162fig2.jpg" /> 図2：ダンと肺凍結切片における微小転移の検出LM8。 10月組み込み肺組織の切片を加湿チャンバー内で24時間37℃でのX-Gal染色液中でインキュベートした後、核ファーストレッドで対比染色した。矢印はダン-lacZを（B）またはLM8-lacZの細胞（D）を注射したマウスの肺切片において認識微小転移巣を指す。 Micrometastasesは非形質ダン（A）またはLM8セル（C）を注射したマウスの肺切片では検出できなかった。スケールバーは0.1mmを示す。

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特別な利害関係は宣言されません。

Dunn/LM8マウスOSモデルでここに示された結果は、肺組織のin-situ灌流/固定でのlacZ-タグ腫瘍細胞のX-gal染色を組み合わせて新たに確立された方法の力を発揮。 2つの技術のこの組み合わせは、単一細胞レベルに微小転移病変の高感度検出を停止でき、また、肺の表面（ 図1）と同様の肺切片における（ 図2）macrometastasesの可視化を向上させます。 X-gal染色も自然なので、血液と組織関連の色の他の臓器のin-situ灌流/固定で（マイクロ）転移の検出がわずかに肺以外の組織における転移巣の検出能を向上させることができますがこれらの器官13。灌流が別の臓器に向けられている場合であっても、肝臓を例えば 、血液の除去は部分的にしかコントラストベットを改善するであろう〜だと思うX-gal染色や臓器の自然な色。しかし、この方法はlacZをタグ付きの腫瘍細胞の任意のタイプに適用され、飛躍的に休眠中の単一セルの微小転移のレベルに肺転移の検出能を向上させ、ダウンマクロおよび微小転移の簡単で信頼性の高い定量が可能になります。この方法の限界及びルシフェラーゼおよび蛍光タンパク質を含むレポーター遺伝子に基づいている他のすべての技術は、導入遺伝子の発現の安定性である。 図2dに示すように、macrometastatic病巣内の全ての腫瘍細胞は、β-ガラクトシダーゼ活性の不足を示す青色に染色されています。これは、壊死に関連するかもしれないが、それは可能性が高く、導入遺伝子発現の損失によるものである。私たちは、ダンとLM8細胞が発現させるためであっても連続的な選択の下lacZおよび他の導入遺伝子のダウンレギュレーションに非常に有効であることを観察した。そこで我々は、マウスK12に最近の研究で切り替えとK7M2と人間HOS、143BとSAOS-2骨肉腫細胞株、in vitroでのすべての維持安定lacZ発現ならびに生体内では、最大時間をかけて100％に。 lacZの発現が保証されて安定したら、この技術は、機構的に転移性病変の根絶15を目指して新たな治療法の開発およびテストを行うためだけでなく、組織の植民地化13のプロセスを調査するために例えば、遺伝子操作された腫瘍細胞を用いた研究で適用することができます、16。さらに、転移性病変の早期検出のために使用される、例えば、PET、（マイクロ）、CTやMRIなどの現在の放射線イメージング技術、改善のためのベンチマークとして機能することができます。異なるトレーサーとの最近のPET研究（未発表）では、我々 は 、in vivoで検出された肺で記述されたプロトコルで、その後、ex vivoで転移が確認された。新しい小動物マイクロCTとの継続的な研究では（SkyScan）我々はこれまでにABですleは0.5ミリメートルとex vivoでダウンして0.3 mmまでのサイズにインビボで肺転移をダウン検出するが、我々は0.1ミリメートルの分解能を目指しています。興味深いことに、これは我々がマクロからのin-situ灌流およびX-gal染色を組み合わせた方法で微小転移を区別するために設定されたサイズ制限があります。これは、再びこの簡単かつ費用対効果の高い技術の感度と有用性を強調している。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> Narketan10（ケタミン） </td><td> VétoquinolAG </td><td></td><td> 100 mg / mlの溶液を</td></tr><tr><td> Xylazin（キシラジン） </td><td> StreuliファーマAG </td><td></td><td> 20 mg / mlの溶液を</td></tr><tr><td> Prequilan（アセプロマジン） </td><td> Fatro SpAの</td><td></td><td> 10 mg / mlの溶液を</td></tr><tr><td>ブルドッグのタイプは、血管クランプを止血小鉗子</td><td>ファイン科学ツール</td><td> 18051から35 </td><td>湾曲した、35ミリメートル</td></tr><tr><td>蓋付きのプラスチックカップ</td><td> Semadeni </td><td> 2988 </td><td>蓋つきの25ミリリットルのコップ</td></tr><tr><td> 5 - ブロモ-4 - クロロ-3 - インドリル-β-D-ガラクトシド（X-Gal）を、粉末</td><td> Axxora </td><td> ALX-582-002-G005 </td><td>店舗-20℃で光保護、N-ジ-メチルホルムアミドに40 mg / mlで、Nに溶解</td></tr><tr><td> maxの光保護4℃で保存されてpHが7.1に平衡化の基本的な染色液、、。 1ヶ月</td><td>自分で作った</td><td></td><td> 100ミリリットル/ L 10xPBS、1.64 g / LのK3Fe（CN）6、2.1 g / LのK4Fe（CN）6、2ミリリットル/ L 1MのMgCl2、2ミリリットル/ L 10％NP40、1ミリリットル/ Lの10％のナトリウムデオキシコール酸</td></tr><tr><td> PolyFreezeの埋め込みミディアム</td><td> Polysciences社、スイスの代理店：Brunschwig </td><td> 19636から1 </td><td></td></tr><tr><td>埋め込み型</td><td>ポリサイエンス</td><td> 18646A-1 </td><td></td></tr><tr><td>ライカCM1850UVクライオスタット</td><td>ライカマイクロシステムズ</td><td></td><td></td></tr><tr><td> SuperFrostスライド</td><td>メンツェル</td><td> J1800AMN​​Z </td><td></td></tr><tr><td>核ファストレッド - 硫酸アルミニウム溶液</td><td>課クロマヴァルデック社＆CO KG DISTributor：Medite </td><td> 84-0241-00 </td><td></td></tr><tr><td>免疫マウント</td><td>サーモエレクトロンは、スイスの代理店：Histocom </td><td> 9990412 </td><td></td></tr></tbody></table> 表1試薬および器具。

improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and x-gal staining of lacz-tagged tumor cells

転移は、がんの種類やがん研究の結果として、主な焦点の大半の死亡の主な原因である。しかし、放射線イメージングおよびそれらの治療根絶の成功により、微小転移の検出が制限されたままです。 動物モデルでは、がん研究のための貴重なツールが1であることが証明されているが、微小転移のモニタリング/可視化は、潜在的に臨床試験2で残念な結果につながる前臨床試験で転移拡散の挑戦と不正確な評価のままです。その結果、最終的にダウンして正常組織における単一細胞レベルへの転移の再現性と信頼性の高い検出を可能にするための方法を洗練することに大きな関心がある。主な焦点は、したがって、マイクロコンピューター断層撮影（マイクロCT）、陽電子放射断層撮影（PET）、生物発光や蛍光イメージングのようなin vivoでの腫瘍細胞の検出を可能にする技術は、オンになっている&lt;&gt; 3,4（商標）。我々は現在、別の骨肉腫モデルにおける原発腫瘍の増殖や転移の in vivoモニタリングのために、これらの技術を最適化している。これらの技術のいくつかは、定量PCR 5、6、FACSまたは組織学的染色の異なるタイプのような古典的な方法の横に転移の ex vivo での解析に使用することができます。ベンチマークとして、我々は発色基質5 -ブロモ-4 -クロロ-3 -インドリル-β-Dを代謝する細菌酵素β-ガラクトシダーゼをコードするlacZ遺伝子と本研究では、腫瘍細胞の安定なトランスフェクションまたは形質導入を確立している不溶性の藍色染料7〜-ガラクトピラノシド（X-Gal）を、ここに示すように、単一細胞レベルにマウス組織を ex vivoでの腫瘍細胞の高感度かつ選択的な組織化学ブルー染色をダウンできます。これは、新規性を評価する研究で転移8の正確な検証を可能にする機器を集中的に低コストではないツールですticancer療法9-11。 X-gal染色の制限要因はよく血管化組織の血液関連赤い染色をEGに低いコントラストである。肺組織では、この問題はその場での肺血流、最近ベルジッヒら 12血液からそれらをクリアすると下にその場でこれを修正して埋め込 ​​むことが麻酔下のマウスの肺を灌流することによって確立された手法によって解決することができます気管を通じてインフレ。この方法はまた、肺の崩壊を防ぎ、それによって組織学的分析のために組織の品質を向上させる機能的な肺胞の形態を維持しています。そこで、本研究では 、lacZ 発現腫瘍細胞と肺組織のin-situ灌流および固定のX-gal染色の組み合わせを活用して新しいプロトコルを記述します。この洗練されたプロトコルは、肺内の単一の転移細胞の高感度検出を可能にし、A〜Dの最近の研究で私たちを有効にもともと14非転移性であることが記載されたマウスモデル13に&quot;休止状態&quot;肺の微小転移をetect。

Watch this Scientific Journal Video about Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tum

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

本研究で報告された新たなプロトコルが組み合わせによるマウスの単一細胞の解像度で肺転移の選択的検出を可能に<em&gt;のin-situ</em&gt;肺血流との固定とX-Gal染色<em&gt; lacZを</em&gt;タグ付きの腫瘍細胞。

組み合わせによるマウスのシングルセル解像度で肺転移の可視化の向上<em&gt;のin-situ</em肺組織とのX-gal染色の&gt;灌流<em&gt; lacZを</em&gt;タグ付き腫瘍細胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

improved-visualization-lung-metastases-at-single-cell-resolution-mice

Research

JoVE Journal

Medicine

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

17.2K ビュー.  Balgrist University Hospital, Zurich. 本研究で報告された新たなプロトコルが組み合わせによるマウスの単一細胞の解像度で肺転移の選択的検出を可能にのin-situ肺血流との固定とX-Gal染色 lacZをタグ付きの腫瘍細胞。

ビデオ: Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

孤立の方法<em&gt;エクスビボ</emラットモデルにおける&gt;肺灌流：ラットEVLPプログラムの開発の教訓

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

医学

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 ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

定量的リアルタイムPCRによるマウス乳腺腫瘍モデルにおける肺転移の評価

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

最大急性非致死肺損傷における急性肺損傷の結果のオープン気管切開胃酸吸引マウスモデル

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

疾患モデル作製のひと 3 D 肺組織培養 (3 D-Ltc) の生成

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

平滑筋細胞系譜トレース マウスの高度な動脈硬化病変における細胞構成の定量的解析

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

直接気管内リポ多糖刺激によるマウスの急性肺損傷の誘導

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

正常母性陰圧換気 ex situ 肺灌流:肺機能と代謝の評価

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

循環器死後の管理された提供における腹部正常局所灌流と組み合わせた肺の急速な回復の調達

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

アガロース包埋ビブラトーム切片を用いた革新的な肺イメージング

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

著者スポットライト:アガロース包埋肺組織切片を用いた空間的タンパク質発現の研究 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

マルチプレックス蛍光イメージングのためのアガロース包埋肺組織の自動ビブラトーム切片化

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...