我々は、一般的な議論のための博士マイケルシモンズ、博士キープハウク、そのラボの両方のメンバーが、プロトコルの初期の試験のためにケリー博士スティーブンに感謝します。 この作品は、NIHの助成金HL087513とP01 HL094374によってサポートされています。

1。出発前の準備<ol><li>それぞれ、血管拡張バッファー（PBS中4mgの/ Lのパパベリン+ 1グラム/ Lアデノシン）、またはPBS中4％パラホルムアルデヒド（PFA）で圧力灌流装置の各側面を埋める。 </li><li> 1:100ヘパリン（5000U/mlストック）の0.1ミリリットルでそれを充填し、上ベベルと〜120度の角度に針を曲げて1/2ccインスリンシリンジ（恒久的に接続されている29Gと½ &quot;針）を準備が増加します。 0.3ミリリットル飽和KCl溶液で満たされた1 mlのシリンジ（26G½ &quot;針付き）と同じです。 </li></ol> 2。心臓を露出し、大動脈をcannulating <ol><li>選択した麻酔薬を使用して、マウスを麻酔。 （私たちは、ケタミン/キシラジン混合物の過剰使用：130 mg / kgのケタミンと生理食塩水の8.8 mg / kgのキシラジンのIP注射）を</li><li>アップ解剖トレイ、腹側に麻酔マウスをピン。正中切開で腹腔を開き、皮膚を撤回臓器を公開します。後部下大静脈（PVC）の領域を露出させる一方の側に腸に移動します。 </li><li> PVCにヘパリン溶液を注入します。あなたが針を抽出するように、漏れを防止し、PVC壁の血栓やアザラシまで数秒のためにそれを保持するために綿棒で針の穴をカバーしています。マウスの循環を通して分散させるためにヘパリン2-3分待ちます。 </li><li>あなたは心臓の鼓動を観察することができるように横隔膜と胸郭を解剖。徐々に心臓の逮捕まで、PVCのKCl溶液を注入します。 </li><li>ダイアフラムの領域前方をそのまま残し、ダイヤフラムと消費税マウスの後部の下にあるすべての臓器を削除します。心臓への近位部は、後続のステップで見つけることは容易であるので、絞りの近くにPVCを切断するように注意して、ダイヤフラムを取り外します。 </li><li>大動脈の切り口を探します。大動脈の下に6から0編組絹縫合糸の1つの長い長さ数ミリメートルあちこち前方に置きメートル縫合糸は、バック自体に倍増されるようにカット終了。大動脈下縫合の2個があるので、半分にこの長い縫合糸をカットします。大動脈（図1A、B）の切断端に血管カテーテルを挿入し、代わりに血管カテーテルを保持し、漏れから大動脈内の任意の背圧を防止するために、ダブルノットで各縫合糸を結ぶ。 </li></ol> 3。血流とMicrofil注入<ol><li>圧力灌流装置（図2）に血管カテーテルを接続し、100から110 mmHgの運転圧力に灌流装置をポンプで血管拡張バッファ（図1C）で血管を灌流を開始します。バッファは、液体を確実にすることによって、coronariesを通して灌流されていることを確認しダブルは、PVCから終了します。少なくとも3分間、またはPVCを出る流体がクリアされるまで灌流し続けています。 （灌流しながら次のステップに進みます。） </li><li>リブとピンバック（または削除）心臓を露出させる胸郭を解剖。一度公開され、CAである心はガーゼのバッファーに浸漬した部分から心臓にバッファの滴を絞って乾燥させないreful。大動脈弓を公開するために胸腺を離れてオフにします。流体がcoronariesを通してではなく、これらの大きい、低抵抗血管（図1Dの）を介して流用されていることを確認するために6から0まで編んだシルク糸を使用して、3つの主要な大動脈の枝を連結。 </li><li> 15分間固定して心臓を灌流し、少なくとも2分間血管拡張バッファーですすぎます。一方、注射後の心臓（図1E）の外に漏れるのMicrofilを防ぐために両方の前部静脈Cavaeを連結。場所は、PVCと大動脈周囲の縫合糸が充填後まで締めないでください。 </li><li> Microfilを準備します（試薬の表で指定された）と1 mlの注射器にロードします。カテーテル（Microfilシリンジに潅流チューブから切り替えたときに気泡の導入を防ぐために）をカバーするのに十分な水で解剖トレイを記入してください。血apを切断カテーテルからparatusと準備Microfilシリンジを接続します。 </li><li>冠状動脈の良好な充填が（図1F、3A）明らかになるまで、大動脈にMicrofilを注入：動脈は、最初に記入し、その後MicrofilはMicrofilの色を持つ組織のフラッシュのように毛細血管に &quot;流出&quot;します。それは小型船から出てくるように一回静脈側では、Microfilの疎水性の性質は、それが最初に独立した球体として表示されます。連続した列が静脈を一杯になるまでMicrofilを注入し続けています。 Microfilは、血管内の連続であるときに完全な充填が明らかとなり、それがPVCを介して終了します。 </li><li>充填が完了した後、すぐに以前の船舶のうちMicrofilを絞ってから、心臓組織の弾性の性質を防止するためにPVCと大動脈の周囲に配置された縫合糸を締めます。 </li><li> （解剖トレイから水に浸した濡れたガーゼで心臓をカバーMicrofilが重合されるまで）は、乾燥し、それが室温で約1時間放置を防ぐことができます。そのような心の奥に満ち船舶の早期ビューを取得しようとして心を持ち上げたり、回転などの重合プロセスの間、心臓の任意の外部からの圧力を避ける。これはMicrofilで改行を引き起こし、心のより弾性領域にいくつかの血管からMicrofilを絞ることができます。 </li><li>心臓を取り出して、4℃で一晩4％PFAでそれをポスト修正℃にその後、4℃、70％エタノールに保存心臓血管系は現在、μCTイメージングのための準備ができています。 </li></ol> 4。代表的な結果効果的にMicrofilで灌流されている船舶は、船舶全体の連続的な、切れ目のないMicrofil（図3A）を持ちます。冠状血管の充填の程度は、目で判断することができる。静脈epicardially 18に配置され、簡単に（図3A、矢印）が観察できます。動脈、これは、18以上の心筋が、心臓の表面（図3A、矢印）からも見ることができます。心臓組織は、毛細血管の非常に高い密度を有し、したがって、ときに毛細血管塗りとしてキャピラリーフィリングにも明らかである、心臓の組織がMicrofilの色（図3A、スター）でフラッシュします。このように、埋めるために失敗した血管網が原因Microfilの欠如（図3B、C）に顕著になります。 Microfilの疎水性の性質は、それ自体に収縮充填容器内の &quot;改行&quot;を引き起こすことになりますので、Microfilの不連続性（図3Bのアスタリスク）が頻繁に表示されます。血管内の圧力は、心臓から血管の出口点の適切な提携オフを通して維持されている場合、これらの &quot;改行&quot;は削減することができます。他の不連続性がmicrofil内の気泡によって発生することができます。灌流APからの切り替え時の空気の導入を防ぐために、血管カテーテルが完全に水に沈めていることを確認してください<html> Microfilシリンジにparatus。気泡が導入されている場合は、それはしばしばバブルが冠状血管の通って押し出されたまでMicrofil灌流を続けることによって、単に削除することができます。 血管床の一部が（図3B、矢印）がブロックされている場合、血管のネットワークは完全に埋めることはできません。ヘパリンは血栓の形成を阻害しながら、時折閉塞はまだ手順を開始する、またはその他の未知の要因に起因する前に起因する不完全なヘパリン血流に発生する可能性があります。閉塞が発生した場合は、血管の塗りつぶしを完了するには、閉塞を外れないための方法は、我々の知る限り、ありません。少なすぎる圧力はMicrofilは、すべての血管床と毛細血管網（図3C）に強制されないので、充填時に使用されている場合、不完全な充填にも発生する可能性があります。逆に、あまりにも多くの圧力が毛細血管周囲の組織（図3D）にMicrofilバーストと浸出する可能性があります。 files/ftp_upload/3740/3740fig1.jpgの &quot;alt =&quot;図1 &quot;/&gt; 図1。 Microfil灌流スキームの概要 。 （A）大動脈とPVCは、ダイアフラムの約レベルでカットされます。 （B）上行大動脈は、血管カテーテルでカニューレ挿入されています。 （D）大動脈弓からの3つの主要な枝が連結されている間（C）血管拡張バッファは、（図示せず）の圧力灌流装置によって駆動され、血管を通って灌流されています。両方の前部静脈のカヴァが連結されている間（E）4％PFAはcoronariesを通して灌流されています。それは塩化ビニル（PVC）から出るが観察されるまで、（F）注射器を使用して、Microfilはcoronariesを通して灌流されています。 <img src="/files/ftp_upload/3740/3740fig2.jpg" alt="図2"/> 図2。灌流装置2つの三角フラスコ、血管拡張バッファまたは4％PFAのどちらかで満たされた各々が、参加し、彼らのsidearmsに接続されたチューブを介して加圧されています。システムは、ポンプマニュアルを加圧されているバルブ、圧力計、圧力のモニタリングとメンテナンスを可能にするためにフラスコのいずれかに接続されています。小さなチューブはゴム栓を介して上下に各フラスコ内の流体に拡張します。圧力はこれらの小さなチューブより各フラスコからsidearmsポンプ流体から入力します。チューブは、一度に1つのフラスコから流れるように流体ができます活栓にマージします。 <img src="/files/ftp_upload/3740/3740fig3.jpg" alt="図3"/> 図3。 Microfilled心をサンプリングします 。 （A）も満たされる船舶は、（もしあれば）Microfilで改行をいくつか持っているし、心臓組織はいっぱい毛細血管（スター、およびCと比較して）によるMicrofilの帯びた色になります。両方の動脈（矢印 - 左前下行枝）と静脈（矢印 - 左冠状静脈）は心臓の表面を介して表示されます。 （B）microfilで休憩（アスタリスク）と同様に完全にマイクを防止するいくつかの血管の閉塞による心浸透をrofil。ブロックされた血管は血液が灌流プロセス中にフラッシュされていないとして、赤（矢印）のままです。 （C）不完全に満たされた血管と心臓。組織がMicrofilが毛細血管に浸透しなかったことを示す、Microfilの黄色の色で撮影していないことに注意してください。 （D）心のどこMicrofilが周囲の組織（矢​​印）に漏れることを引き起こして、充填中の毛細血管が破裂。

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+density,+remodeling,+and+VEGF-A+expression+differ+widely+between+mouse+strains.">Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains.</a> Physiol. Genomics. 30, 179-191 (2007).</li><li>Behm, C. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Endothelial+Vascular+Cell+Adhesion+Molecule-1+Expression+and+Inflammatory+Cell+Recruitment+During+Vasculogenesis+and+Ischemia-Mediated+Arteriogenesis.">Molecular Imaging of Endothelial Vascular Cell Adhesion Molecule-1 Expression and Inflammatory Cell Recruitment During Vasculogenesis and Ischemia-Mediated Arteriogenesis.</a> Circulation. 117, 2902-2911 (2008).</li><li>Carr, C. L., Lindner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+perfusion+imaging+with+contrast+echocardiography.">Myocardial perfusion imaging with contrast echocardiography.</a> Curr. Cardiol. Rep. 10, 233-239 (2008).</li><li>Leong-Poi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Endogenous+and+Therapeutic+Arteriogenesis+by+Contrast+Ultrasound+Molecular+Imaging+of+Integrin+Expression.">Assessment of Endogenous and Therapeutic Arteriogenesis by Contrast Ultrasound Molecular Imaging of Integrin Expression.</a> Circulation. 111, 3248-3254 (2005).</li><li>Villanueva, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+Targeted+to+Intercellular+Adhesion+Molecule-1+Bind+to+Activated+Coronary+Artery+Endothelial+Cells.">Microbubbles Targeted to Intercellular Adhesion Molecule-1 Bind to Activated Coronary Artery Endothelial Cells.</a> Circulation. 98, 1-5 (1998).</li><li>Wei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Myocardial+Blood+Flow+With+Ultrasound-Induced+Destruction+of+Microbubbles+Administered+as+a+Constant+Venous+Infusion.">Quantification of Myocardial Blood Flow With Ultrasound-Induced Destruction of Microbubbles Administered as a Constant Venous Infusion.</a> Circulation. 97, 473-483 (1998).</li><li>Beckmann, N., Stirnimann, R., Bochelen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Magnetic+Resonance+Angiography+of+the+Mouse+Brain:+Application+to+Murine+Focal+Cerebral+Ischemia+Models.">High-Resolution Magnetic Resonance Angiography of the Mouse Brain: Application to Murine Focal Cerebral Ischemia Models.</a> Journal of Magnetic Resonance. 140, 442-450 (1999).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+MR+angiography+of+intratumoral+vasculature+using+a+novel+macromolecular+MR+contrast+agent.">3D MR angiography of intratumoral vasculature using a novel macromolecular MR contrast agent.</a> Magnetic Resonance in Medicine. 46, 579-585 (2001).</li><li>Nezafat, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B1-insensitive+T2+preparation+for+improved+coronary+magnetic+resonance+angiography+at+3+T.">B1-insensitive T2 preparation for improved coronary magnetic resonance angiography at 3 T.</a> Magn. Reson. Med. 55, 858-864 (2006).</li><li>Wagner, S., Helisch, A., Ziegelhoeffer, T., Bachmann, G., Schaper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+angiography+of+collateral+vessels+in+a+murine+femoral+artery+ligation+model.">Magnetic resonance angiography of collateral vessels in a murine femoral artery ligation model.</a> NMR in Biomedicine. 17, 21-27 (2004).</li><li>Cochet, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MR+angiography+and+velocity+measurement+in+mice+coronary+arteries+at+9.4+T:+assessment+of+coronary+flow+velocity+reserve.">In vivo MR angiography and velocity measurement in mice coronary arteries at 9.4 T: assessment of coronary flow velocity reserve.</a> Radiology. , 254-441 (2010).</li></ol>

マウスは、ワシントン大学の動物実験及び利用委員会と米国国立衛生研究所（NIH出版番号85から23で公開実験動物のケアと使用のためのガイドに従い、承認された方法で処理された）1996を改訂しました。

心臓組織は非常に高い代謝需要を持っているため、冠状血管系によって提供される血液から栄養と酸素の安定供給を必要とします。血管の狭窄や閉塞が原因で冠状動脈の機能を低下させる冠状血管の疾患は、組織低酸素および虚血につながると、心筋梗塞や心臓の筋肉への取り返しのつかない損傷の危険性が影響を受けた患者を置くことができます。これらの血管の病気の状態の理解が必要?...

<html><table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> 1 mlのシリンジ</td><td> <a href="http://www.bd.com/" target="_blank">ベクトン·ディッキンソン</a> </td><td> BD-309602 </td><td></td></tr><tr><td>恒久的に接続されている29G½ &#39;針1/2ccインスリンシリンジ</td><td> <a href="http://www.bd.com/" target="_blank">ベクトン·ディッキンソン</a> </td><td> BD-309306 </td><td></td></tr><tr><td> 2 &quot;×2&quot;ガーゼパッド</td><td> Med101store.com </td><td> SKU 2208 </td><td></td></tr><tr><td> 24G¾ &quot;Angiocath IVカテーテル</td><td> <a href="http://www.bd.com/" target="_blank">ベクトン·ディッキンソン</a> </td><td> BD-381112 </td><td></td></tr><tr><td> 26G½ &quot;ゲージの針</td><td>痩せ細った &quot;&gt;ベクトン·ディッキンソン</td><td> BD-305111 </td><td></td></tr><tr><td>アデノシン</td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">シグマ</a> </td><td> A9251 </td><td>血管拡張バッファのPBSで1グラム/ L（パパベリン付） </td></tr><tr><td>斜めグレーフェ鉗子</td><td> <a href="http://www.finescience.com/" target="_blank">ファイン科学ツール</a> </td><td> 11052から10 </td><td></td></tr><tr><td>綿棒のアプリケーター：6 &quot;非滅菌</td><td> <a href="http://www.cardinal.com/" target="_blank">カーディナルヘルス</a> </td><td> C15055-006 </td><td></td></tr><tr><td>湾曲した外科用はさみ</td><td> <a href="http://www.finescience.com/" target="_blank">ファイン科学ツール</a> </td><td> 14085から09 </td><td></td></tr><tr><td>解剖立体と光源</td><td> <a href="http://www.nikon.com/" target="_blank">ニコン</a> </td><td> NA </td><td> NA </td></tr><tr><td>解剖トレイ、29.2 x 7.5インチ</td><td> <a href="http://www.coleparmer.com/" target="_blank">コール·パーマー</a> </td><td> YO-10915から12 </td><td>マウスを下に固定するためのタールで満たされた</td></tr><tr><td>ファイン曲がり鉗子</td><td> <a href="http://www.aesculapusa.com/" target="_blank">Aesculap</a> </td><td> FD281R </td><td>必要がある2つの</td></tr><tr><td>ヘパリン、5000 U / mlストック</td><td> <a href="http://www.apppharma.com/" target="_blank">APPファーマシューティカルLLC</a> </td><td> NDC 63323-047-10 </td><td>水で100倍希釈</td></tr><tr><td>塩化カリウム</td><td> <a href="http://www.fishersci.com/" target="_blank">フィッシャー</a> </td><td> P217 </td><td> H2Oの飽和溶液</td></tr><tr><td>ケタミン（Ketaset）、100 mg / mlストック</td><td>フォートドッジ、オーバーランドパーク、カンザス州、アメリカ合衆国</td><td> NDC 0856-2013-01 </td><td> 0.9パーセントsのキシラジンで、130 mg / kg体重のように混合アリーン</td></tr><tr><td> Microfil </td><td> <a href="http://www.flowtech-inc.com/" target="_blank">フローテック</a> </td><td> MV-122（黄色）。他のカラーオプションも用意されています。 </td><td>硬化剤の体積10％、重量比で1:1に混合します。それが十分に混合であることを確認するためだけに注入し、渦の前準備</td></tr><tr><td>非滅菌縫合糸：6-0、編んだシルク</td><td> <a href="http://www.harvardapparatus.com/" target="_blank">ハーバード装置</a> </td><td> 723287 </td><td></td></tr><tr><td>パパベリン</td><td> <a href="http://www.americanregent.com/" target="_blank">アメリカのリージェント株式会社</a> </td><td> NDC 0517-4010-01 </td><td>血管拡張バッファのPBS中4mgの/ L（アデノシン付） </td></tr><tr><td>パラホルムアルデヒド</td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">シグマ</a> </td><td> P6148 </td><td> 4％の溶液として調製し</td></tr><tr><td>灌流装置</td><td></td><td></td><td>図2を参照してください。 </td></tr><tr><td>春のはさみ</td><td> <a href="http://www.finescience.com/" target="_blank">ファイン科学ツール</a> </td><td> 15018から10 </td><td></td></tr><tr><td>キシラジン（Anased）に20 mg / GL株式</td><td> <a href="http://www.lloydlab.com/" target="_blank">ロイドラボ</a> </td><td> NADA＃139から236 </td><td> 0.9％生理食塩水のケタミンで、8.8 mg / kg体重のように混合</td></tr></tbody></table>

retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging

血管系の可視化は、さまざまな病気の状態を理解するためにますます重要になっています。いくつかの技術は、撮像血管系に存在するが、いくつかは、小型船1,2が含まれています解像度を拡張しながら全体としての血管網を可視化することができます。さらに、多くの血管の鋳造技術は、サンプル3-5のさらなる解析を防ぐために、周囲の組織を破壊する。これらの問題を回避する一つの方法は、マイクロコンピューター断層撮影​​（μCT）です。 μCTイメージングは、解像度が&lt;10ミクロンでスキャンすることができます血管網の3次元再構成を作り出すことができるであり、その後の分析（例えば、組織学および形態計測）6月11日のために無傷の組織を残します。しかし、ex vivoで μCT法によるイメージング血管は血管が放射線不透過性化合物で満たされている必要があります。このように、μCT画像によって生成された脈管構造の正確な表現は時に​​偶発的です。信頼性の高い容器の充填が完了しました。このプロトコルでは、我々は、μCT画像の準備のためにマウス冠状血管を充填するためのテクニックについて説明します。 二つの支配的なテクニックは、冠状動脈血管を埋めるために存在する：in vivoでの挿管とランゲンドルフ灌流システム15から17を経由して大動脈（または大動脈弓から分岐）12から14、またはex vivoでの逆行性血流を介して。ここでは、特にすべての船舶の充填を確認するように設計されている生体大動脈カニュレーション法で説明しています。我々は、すべての毛細血管だけでなく、血管網の動脈および静脈の両側を埋めるために、最小血管を灌流することができますMicrofilと呼ばれる低粘度の放射線不透過性化合物を使用しています。血管は加圧された灌流システムを用いた緩衝液で灌流し、Microfilで満たされている。そのMicrofilが小さい高抵抗血管を埋めるようにするために、我々は、大規模なブランチemanatinを連結coronariesにMicrofilをそらして大動脈からグラム。一度の充填には、いくつかの船のMicrofilを絞り出しから心臓組織の弾性の性質を防止するために、完了したら、我々はすぐに充填した後にアクセス可能な主要な血管の出口点を連結。動脈、毛細血管、静脈同様に - したがって、我々の手法は、完全な冠状血管網の可視化を可能にする、完全な充填および充填剤の最大保持のために最適化されています。

Watch this Scientific Journal Video about Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging at JoVE.com

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

冠状血管の可視化は、心血管疾患の我々の理解を進めることが重要です。ここでは、マイクロコンピューター断層撮影​​（μCT）イメージングの準備のために、放射線不透過性シリコーンゴム（Microfil）とマウス冠状動脈血管を灌流する方法を説明します。

マイクロCTイメージングのための準備として、逆行性灌流およびマウスの冠動脈血管系の充填

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

retrograde-perfusion-filling-mouse-coronary-vasculature-as

Research

JoVE Journal

Medicine

32.3K ビュー.  University of Washington. 冠状血管の可視化は、心血管疾患の我々の理解を進めることが重要です。ここでは、マイクロコンピューター断層撮影​​（μCT）イメージングの準備のために、放射線不透過性シリコーンゴム（Microfil）とマウス冠状動脈血管を灌流する方法を説明します。

ビデオ: Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

X線CTを用いたマウス肥満モデルにおけるセグメンテーションおよびFATボリュームの測定

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

医学

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

マイクロコンピュータ断層撮影イメージング用Microfilで虚血後肢と血管系の灌流に浸透ドラッグデリバリー

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

マイクロコンピュータ断層撮影とヌードマウスへのヒト脂肪注入の生存率の評価

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

腫瘍の開発の評価のためのマイクロコンピュータ断層撮影を使用し、肺癌のマウスモデルでの治療に対する応答のフォローアップ

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

直接脳血栓塞栓を可視化するための複合近赤外蛍光イメージングおよびマイクロコンピュータ断層撮影

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

断層レントゲン写真撮影スキャンを計算非ゲートの冠動脈石灰化を識別します。

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

小動物陽電子放出断層撮影/コンピュータ断層撮影における連続的な血液サンプリングにより、動脈入力機能の測定が可能

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

慢性疾患を有する大型哺乳類心臓モデルのコントラスト増強マイクロコンピュータ断層撮影イメージングのための組織調製技術

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

ダクト:3D肝臓解析のためのマイクロコンピュータ断層撮影に続く二重樹脂鋳造

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

コンピューター断層撮影ベースの3次元可視化のサンプル調製

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...