我々は、肺の鋳造を支援博士Adlah Sukkar、MDの仕事を承諾します。この作品は、NHLBI、HL088005によって資金を供給されています。

ラット上で実行されるすべてのプロトコルは、ジョンズ·ホプキンス大学の動物実験委員会及びNIHガイドラインに従って承認されている。可能な限り、動物を外科手術部位の汚染を最小限に抑えるために手術領域から別の領域に整形処理されるべきである。 1。麻酔/鎮痛<ol><li>イソフルラン3パーセントを注入誘導チャンバー内の、場所のラット（ハーラン、インディアナポリス、インディアナ州のSprague Dawley雄ラット、125〜150グラム）。 </li><li> 3パーセントイソフルラン麻酔とノーズコーンと人工呼吸器に接続されている手術·ボード上に麻酔をかけたラットを置きます。すべてのプロシージャのための無菌技術を使用しています。無菌手術領域を確保するためにラットをドレープ。 </li></ol> 2。挿管<ol><li>外科用テープを使用して仰臥位で肢を固定。 </li><li>ノーズコーンからラットを外し、パッドを入れられた鉗子で舌を伸ばす。 </li><li>ガイドとして鈍い金属スタイレットと14ゲージintracathを使用しています。に後ろにスライドさせます気管にngue、。 </li><li>気管内に白いプラスチックintracathを残し金属スタイレットを取り外します。ラットが呼吸していると空気が管を通って流れていることを確認します。 </li><li>カテーテルに直接アダプタにノーズコーンを交換して、人工呼吸器（; 8ミリリットル/ kgを一回換気量、齧歯ベンチレーターモデル683、ハーバード装置、ホリストン、マサチューセッツ州90回/分）にラットを接続します。 </li><li> Puralube（シャインバトラー、オハイオ州ダブリン）目に獣医軟膏を塗る。 </li></ol> 3。開胸術<ol><li>場所ラット右側を下には、外科用テープを使用して付属物を固定する。 </li><li>左側胸郭領域を剃る。 </li><li>小型真空で過剰毛皮を取り外します。 </li><li>ポビドンヨードswabstick（Dynarex株式会社Orangebur、NY）に続いてアルコールで拭くことによって無菌領域を作成します。このプロセスを2回以上（3スクラブの合計）を繰り返します。 </li><li>フィールドの中央に無菌の解剖ハサミまたは滅菌メスで横切開を加えます。 </li><li>リットルを通して解剖鈍ダウンリブに対する組織と脂肪のエアーズ（最後の層は肋骨を覆う薄い膜です）。 </li><li>第3肋間を決定するために肋骨を数える。 </li><li>無菌の45°グレーフェ鉗子を用いて、3回目と4 回目のリブの間に鈍い切開を行います。 </li><li>リブの区切りを挿入して、開いたままにして縫合糸で、肺の完全な可視化機能を備えたオープン·ボイドが優しく場所テープを引き出します。 </li></ol> 4。左肺動脈結紮<ol><li> 90°グレーフェ鉗子で、右手で背中左肺に移動します。 </li><li>デュモンパターン＃5ストレートピンセットを用いて、左手で左肺動脈と気道をつかむ。左肺動脈は気道の上に横たわっています。鉗子は、テーブルに直接垂直であることを確認します。さらに、それは最も遠位の位置（実質に近い）で左肺動脈/左主気管支を拾うために最も効果的である。この左側の操縦にして脇に置いておき換気左肺を押してください。 </li><li>デュモンを使用パターン＃5 45°彼らの自然な境界で左主気管支から左肺動脈を分離するための湾曲したピンセット。この区切り線は、2つの個々の構造の間に薄いと白で表示されます。 </li><li>血管を経由せずに動脈の直下区切ります。スムーズ分離に沿って一緒に開催された鉗子先端をスライドさせます。 </li><li>鉗子の先端が目に見えて左肺動脈と左主気管支を分離するまで続行します。先端のわずかな点は完全にスルーする必要があります。血液は、鉗子の先端に可視化することができるなら、それは完全に通っていないとライゲーションは試みられるべきではありません。一度スペースを通って、左肺動脈は、鉗子の曲線上に横たわっています。この位置で保持します。 </li><li>優しくまっすぐ鈍鉗子グリップ（左手）を放してプレカット縫合（〜2-3インチ、ポリプロピレン縫合サイズ6から0、マイコ医療、ノースカロライナ州ケアリー）の部分をつかむ。 </li><li>左肺動脈とgをあやし湾曲鉗子を開くRABの縫合。優しく鉗子の曲線に対して上方運動で左肺動脈と左主気管支の間の空間を縫合糸を引き出します。 </li><li>慎重小間結び、縫合糸のスニップ、残りは左肺を閉塞縛り付ける。 </li><li> （青モノフィラメント）ポリプロピレンで二回リブ及び縫合糸を保持するために鈍鉗子を使用して閉じるリブ大きさないように注意されて、19ミリメートル、止血に針を（マイコ医療、Cary、NC）を切断3月8日サークル逆に接続されている4から0縫合スキン（のみリブ）。 </li><li>緩い本結びを完成させ、肺を膨らませて、呼気終末陽圧（PEEP、2月5日CMH 2 O）の上に置き、安全な結び目を肺をしっかりと超インフレにすると、縫合糸の残りの部分を切り取る前に、別の完全な結び目を作る。 PEEPから取り外し、肺が崩れないようにするために30秒間可視化する。 5滴BupivicaineをAPP（医薬品、Schaumbur、IL）を適用します。 </li><li>傷口に組織接着剤を配置することによって、皮膚を​​閉じて、途中で戻って使用して一緒に肌をプッシュ綿の先端アプリケータのd。その後、切開部位で24時間毎に8時間で、または動物が正常な活動を再開するまでBupivicaine（2.0 mg / kgの皮下）を得た。このエリアの皮下層が非常に薄いように、それは自分自身で縫合することは容易ではない。組織接着剤が適用されると皮膚が一緒に押されたとき、それはまた、皮下層を閉じます。 </li><li>イソフルランガスをオフにして自主的な動きが戻ってくるまで部屋の空気で1〜2分間ラットを換気し続けています。人工呼吸器から気管チューブを外して、ネズミがそれを削除する前に、自発的に呼吸していることを確認してください。 </li><li>綿棒とモニタの移動およびリカバリに対応した目からPuralubeを拭き取ってください。ブプレノルフィン塩酸塩（0.05 mg / kgを腹腔内、バトラーシャイン、ダブリンOH）を注入します。手術後48時間ごとに12時間の鎮痛薬の配達を続けている。 </li></ol> 5。左頸動脈にカニューレ気管支perfusの大きさを評価するために、左肺動脈結紮後の所望の時点で虚血性左肺のイオンは、大動脈弓に左頸動脈を介して標識した微小球を注入します。 1月2日、上記のようにラットを準備します。 <ol><li>首に沿って正中線をカットし、気管と左頸動脈（マイクロスフェア注射部位）を明らかにするために解剖鈍らせる。 </li><li> 25グラムの針、4ウェイストップコック、1mlシリンジに接続して、容器内にPE20チューブ（Becton Dickinson社製、MDをスパークス）のヘパリン化生理食塩水、鈍先端で満たさカテーテルを挿入します。 </li><li> 30秒間水を超音波処理で、ミクロスフェアの代わりボトル（インビトロジェン社、ユージーン、オレゴン州15μmで深紅のポリスチレン蛍光ミクロスフェア、1×10 6球/ mlとなる）。 </li><li>ボトル、渦を取り外して、20グラムの針を介して1ミリリットルハミルトンガラスシリンジ（ハミルトン社、リノ、ネバダ州）に0.5ミリリットル（500,000マイクロスフェア）を策定する。 </li><li> 4方活栓にハミルトンシリンジを取り付け、シリンジポンプを用いて微小球を吹き込む（速度：500μL/分;魔神プラス、​​ケント·サイエンティフィック、Torringt）CT、上の。 </li><li>を500μl/分でheparized生理食塩水1mlでハミルトンシリンジとフラッシュ装置を取り外します。 </li><li>完全な胸の開胸を行い、下大静脈を切断することによってラットを放血させる。 </li><li>左肺および関心のある他の組織を削除します。 </li><li>組織からミクロスを抽出するには、放血後のラットの左肺全体を取ると2M KOH（4-6 ml）に入れてください。 55℃の水浴中でプレイスと組織消化のために一晩のままにしておきます。ビーズ、ボルテックス（10秒）、遠心（; 20℃で10分間2000 rpm）を洗浄するためにTween80を（0.25％）を追加します。上清を取り除き、2 - エトキシエチル（1ml）を追加し、ボルテックスし、1時間放置する。渦サスペンション、遠心（2,000 rpmで、（20℃10分間）日立F-2500蛍光分光光度計を用いて測定キュベットで蛍光、場所を含む2 - エトキシエチルアセテート、水層（励起612/emission 618を削除します。 ; DIGILAB、ホリストン、マサチューセッツ州）。 </li></ol>

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試験したすべての種で左肺動脈の結紮は、虚血性の肺の堅牢な全身性血管新生につながる。我々はラットモデルにおける外科的アプローチの詳細を提示している。我々の結果は、血管の鋳造、病理組織によって作り出され、in vivo標識に気管支動脈が増殖し、肺実質を灌流することを示す。したがって、気管支血管新生のメカニズムが慢性肺血栓塞栓症の人間の条件に匹敵する動...

肺における全身の血管新生は、よく認識されています。例えば、喘息2、間質性肺線維症3、癌4、慢性肺血栓塞栓症5で全身血管系および肺実質肺増殖を取り囲み、侵入などの疾患の状態にあります。しかし、全身のこの差の活性化ではなく、肺循環を研究するための動物モデルがいくつかあります。おそらく成人哺乳類の肺における全身の血管新生の最も再現性のモデルは、慢性的な肺動脈虚血を誘発した後に発生するということです。 5月7日 、ヒトの左肺 ​​動脈閉塞に対する応答は、8匹の犬、豚9、10羊、モルモット11匹のラット1、12、13、およびマウス14が気管支動脈と同様に肋間動脈の急速な増殖である。全身neovasculariの責任のメカニズム肺虚血後の肺のzationはほとんど不明であり、広く研究されていない。左肺動脈閉塞後のラットにおける気管支血管新生の時間経過を注意深くウェイベル1の組織の仕事を説明してきた。ラットでこの仕事拡張、我々の研究室では、このプロセスの重要な成長因子ならびに肺におけるこの新生血管の生理学的結果の両方に焦点を当てている。結果は、CXCケモカインCINC-3は初期の虚血後の上昇とCXCR2に対する中和抗体を用いてラットを治療する、CINC-3受容体は、血管新生13を減衰させます。されることを示す14日間肺虚血発症後新たに設立された気管支血管系が大幅に増加した蛋白質の透過性15で異常であることが示された。左肺機能が拡散能力と肺気量の減少が15減少し、通常の上映はありませんでした。新生血管は、共同があるかもしれませんが慢性肺虚血時の肺組織の保全にntributed、それは正常ではないと表示され、肺機能の持続的な減少に貢献するかもしれない。 おそらく、このモデルの最も奇妙な側面の一つは、血管増殖の空間分布に関するものである。虚血による肺実質内の成長因子の放出にもかかわらず、新生血管は比較的大きい上流の気管支動脈に由来する。通常の気管支動脈は、大動脈からの小さな枝のように発生し、気管分岐部における気道ツリーに侵入する。したがって、成長因子は動脈形成の初期段階を誘導するメカニズムは明らかではない。我々は、ラットは、ヒトに類似した血管解剖で、肺虚血時の全身の血管新生の責任のメカニズムを研究するためのユニークな機会を提供することを示唆している。左肺動脈の完全閉塞は、ヒト対象におけるまれなことですが、増加気管支血管分布は同様に何でも肺動脈閉塞16の部位と大きさの患者で誘導されることが表示されます。従って、我々は、ラットの左肺動脈と血管新生の大きさを定量化するための手段を連結するための外科的アプローチの詳細な説明を提供します。

<table border="1"><tbody><tr><td> 試薬： </td><td> 会社 </td><td></td><td></td></tr><tr><td>塩酸ブプレノルフィン、Puralube </td><td>バトラーシャイン</td><td></td><td></td></tr><tr><td> bupivicaine </td><td> APPファーマシューティカル</td><td></td><td></td></tr><tr><td>ポビドンヨードswabstick </td><td> Dynarex株式会社</td><td></td><td></td></tr><tr><td>ポリプロピレン縫合サイズ6-0、3月8日サークルリバースカッティング針</td><td>マイコメディカル</td><td></td><td></td></tr><tr><td> PE20チューブ</td><td>ベクトン·ディッキンソン</td><td></td><td></td></tr><tr><td> 15μmで深紅ポリスチレン標準粒子</td><td>インビトロジェン</td><td></td><td></td></tr><tr><td> 1ミリリットルガラスハミルトンシリンジ</td><td>ハミルトン株式会社</td><td> &lt;/ TD&gt; <td></td></tr><tr><td> 機器： </td><td></td><td></td><td></td></tr><tr><td>魔神プラスシリンジポンプ</td><td>ケント·サイエンティフィック</td><td></td><td></td></tr><tr><td>蛍光分光光度計</td><td> DIGILAB </td><td></td><td></td></tr><tr><td>齧歯類ベンチレーターモデル683 </td><td>ハーバード装置</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 表1特定の試薬 ​​や機器の表。 </td></tr></tbody></table>

angiogenesis in the ischemic rat lung

成人肺は全身気管支動脈と肺動脈を流れる全体の静脈還流の両方によって灌流される。ほとんどの肺の病変では、強化された肺血流の必要性に反応し、強固な血管新生を示す小さい全身の血管系である。肺動脈の閉塞によって誘発される肺血管虚血は人間にだけでなく、いくつかの動物モデルにおける急速な全身の動脈血管新生をもたらすことが示されている。ラットにおける気管支動脈増殖の経時変化の組織学的評価は慎重にウェイベル1で説明したが、新造船のこの組織的な成長のための責任のメカニズムは明確ではありません。我々は気管支血管新生につながるラットに左肺動脈の虚血を誘発する手術の詳細を提供します。血管新生の程度の定量化は、肺内の二つの血管床の存在に起因する新たな課題が発生します。メソッド標識ミクロスフェアの注射に基づく機能血管新生を決定するために提供されています。

血管キャスト：ラットにおける左肺動脈の虚血の影響の結果を図1に描かれている。示す気管支血管系及び28日後に左LPAL気道ツリーの広範な血管のメタクリレートキャストです。このキャストを取得するには、全身の血管系は、下行大動脈への逆行、メタクリル酸混合物（赤）を注射したと気管カニューレを挿入し、白のシリコン系材料を注入した。この血管のキャストは、肺の?...

Watch this Scientific Journal Video about Angiogenesis in the Ischemic Rat Lung at JoVE.com

Angiogenesis in the Ischemic Rat Lung

肺は全身気管支動脈と肺動脈の両方によって灌流される。ほとんどの肺の病変では、堅牢な血管新生を示す小さい全身の血管系である。肺血流の停止は好調気管支血管新生を促進する。我々は気管支血管新生を促進し、左肺動脈の虚血を誘導手術の詳細を提供します。

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 ...

Research

JoVE Journal

Medicine

15.5K ビュー.  Johns Hopkins University. 肺は全身気管支動脈と肺動脈の両方によって灌流される。ほとんどの肺の病変では、堅牢な血管新生を示す小さい全身の血管系である。肺血流の停止は好調気管支血管新生を促進する。我々は気管支血管新生を促進し、左肺動脈の虚血を誘導手術の詳細を提供します。

ビデオ: Angiogenesis in the Ischemic Rat Lung

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

医学

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 ...

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

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

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 ...

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 ...

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

光化学誘起後方虚血性視神経障害のラットモデル

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

ラット異腹部ボリュームにロードされた構成でのハート/シングル肺移植

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

ラット虚血皮膚創傷モデルの実証

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

冠動脈血管新生のインビトロモデル

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

温虚血/再灌流損傷のラット肺移植モデル:転帰を改善するための最適化

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

<meta charset="utf8"></head><body>マウス肺移植における細胞動態のリアルタイムモニタリング

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>著者スポットライト:Intravital Imagingによる肺移植免疫学の進歩

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

移植されたマウス肺の生体内2光子顕微鏡検査

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...