著者は編集支援のために、マイクロCT手術支援のためのイメージング、エドワードTH Yehは、MD、そしてレベッカバートー、博士と彼の技術支援のためのMDアンダーソン小動物イメージング施設のキース·ミシェルに感謝したいと思います。この作品は、アメリカ心臓協会によって部分的にサポートされていました。

1。プライミング浸透圧ポンプ<ol><li>ポンプを準備するとき（ 例えば 、手袋、層流フード）無菌テクニックを使用してください。パッケージングからポンプとフロー司会を削除します。空ポンプを計量し、重量（mg）を記録する。 </li><li>小さな1CCシリンジと鈍ひっくり返さ27G充填チューブを使用することにより、気泡を作成しないように、ゆっくりとポンプを埋める。溶液は、ポンプの上部に達すると、充填停止する。 </li><li>過剰の溶液を拭き取り、いっぱいポンプ重量を量る。ほとんどの水溶液では、mg単位重量はμLのボリュームと同じです。空対充填された重量差は、指定された平均充填容積の90％以上であるべきである。そうでない場合は、シリンジと補充でポンプを避難。あなたが技術的な問題が発生した場合にはhttp://www.alzet.com/products/guide_to_use/filling.htmlでポンプ命令のトラブルシューティングの項を参照してください</li><li>フローmoderatから白フランジを破るためにピンセットを使用してくださいまたは、チューブを曲げたり潰したりしないように注意してください。 3〜4ミリメートル程度カバーし、司会のいずれかの端にP50カテーテルを取り付けます。カテーテルおよび添付司会を埋める、カテーテルの先端部に取り付け、シリンジを残して、シリンジを使用してください。 </li><li>カテーテルは、ポンプと面一になるまでポンプに流れ司会を挿入します。その後、カテーテルの先端からシリンジを取り外します。プライムに6時間（好ましくはO / N）マウスにそれを注入する前に、ポンプの最低37℃のインキュベーター中の滅菌0.9％食塩水またはPBS溶液にポンプを配置します。ポンプ吐出速度は、モデル番号（14日間すなわちモデル 1002 = 0.25μL/時）によって異なります。 </li></ol> 2。マウスで片側後肢虚血を作成する<ol><li> 0.4 L / minの流量で100％O 2で2〜4％の間でイソフルランセットでマウスを麻酔し、depilatoと後肢と背面領域に髪（好ましくは手術前の日）を除去するRYクリーム。 </li><li>手術前に、すべての手術器具を滅菌。誘導麻酔室にマウスを置きます。 2.1節で説明したのと同じ麻酔設定を使用します。 </li><li>マウスが外部刺激に応答しないになるまで待ってから、（1.5から2.0パーセント）イソフルランの連続的な流れを実現しフィットノーズコーンと手術台の上に腹臥位に配置します。 </li><li>切開部位で、麻酔薬の推奨用量（0.25％（&lt;1ミリリットル/ kg）のブピバカイン）を、注入し、70％エタノールに続いポビドンヨード液で肌を扱う。背中（脊柱の側で、肩甲骨領域）上の4〜6ミリメートルの切開を行います。細かい鉗子で皮下膜を分離してから、70％エタノールに続いポビドンヨード液で肌を治療し、大腿内側に向かって膝上のマイクロ手術用ハサミで0.8〜1センチメートル切開を行う仰臥位にマウスを回し、大腿動脈の道に続く。 </li><li>アップトンネル背面の切開に大腿内側から小さなシースチューブ（6 - フランス語）と胴体の側面。 </li><li>腹臥位でマウスを置きます。 PBS溶液からプライミング浸透圧ポンプ/カテーテルを外し、太ももに、シースチューブを通して、カテーテルを導く。 </li><li>背中内側にポンプを置き、6-0プロレン縫合糸（70％エタノールに続いポビドンヨード液で肌を扱う）で逆切開を閉じます。仰臥位で戻ってマウスを置き、シースチューブを取り除く、太ももに切開を介して公開カテーテルを残して。外科テープで脚を固定します。 </li><li>血管系を明らかにするために皮下脂肪組織を分離する。良い血管系を可視化するために、切開を開くためにトラクターを使用しています。 </li><li>ピアース維管束にアクセスするために微尖ったピンセットを使用して優しく膜大腿シース。解剖すると股間近く大腿静脈や神経から大腿動脈を分離し、生じないように注意しながら元cessive出血。 </li><li>大腿分岐（大腿深とepigastrica動脈の起源）の近位大腿動脈2〜3ミリメートルを中断するための場所2 8-0 PROLENE隣接縫合糸を。 </li><li>適切な長さにカテーテルをカットし、物質の標的送達を確保するための浅筋膜へ6-0プロレン縫合でそれを固定し、大腿部の前外側領域にチップを置きます。 6-0実行中の縫合糸で脚切開を閉じます。鎮痛剤の最終的な皮下注射（ブプレノルフィン、0.1 mg / kg体重）を管理、70％エタノールに続いポビドンヨード液で肌を治療し、ケージにマウスを返す。リカバリ中に加熱ランプを使用してください。 </li><li> 30分間の回復期間の後、後肢虚血の作成を確認するためにレーザードップラーイメージングのためのマウスを用意しております。 </li><li>手術後5日間1日2回、動物を監視します。動物の移動度は痛みを分析することが観察されます。ケージ内の廃棄物はインド語として機能します食べるator。動物は痛みや不快感を経験している場合にも、モニター期間中に決定し、二回毎日ブプレノルフィン（0.1mg/kg）の皮下注射を与える。 </li></ol> 3。イメージング試料作製<ol><li> 2.1節で述べたように、マウスを麻酔。仰臥位で手術台の上にマウスを転送します。血管系の凝固を防ぐために、皮下にヘパリン1,000 IUを注入。マウスがノーズコーンに接続されている間、胸腔と下腹部を露出するのに十分な大きさで切開を行います。大動脈腹部大動脈（IAA）を露出するために動物の左側腹部臓器を移動します。下大静脈静脈から大動脈を慎重に分離します。 </li><li>カテーテル挿入時の出血を減らすために、近位IAAを連結。その後、IAAに小さな切開を行います。 IAAに½G鈍針（P10カテーテルに接続されている）27を挿入し、6-0縫合糸で固定します。 </li><li>塗りつぶし220ccをヘパリン化生理食塩水注射し、10％中性（NBF）緩衝ホルマリン。コントラストX線不透過性シリコーンゴム化合物と5ccの注射器を埋める。 MV-希釈剤：1:1.25:0.5の作業比率のMV-硬化剤MV-化合物の3成分を混ぜる。エージェントは、重合前に混合物（Microfil）は、わずか20分の作業時間を持っているので、血管系に注入する直前までMV硬化剤を添加しないことをお勧めします。 </li><li>右心房にカットをすると安楽死を誘導すると、余分な水分の排出を可能にするために、イソフルランの過剰摂取を管理します。針先に生理食塩水を充填したシリンジを取り付け、5分間血管系をフラッシュし始める。すべての血液が血管の外にフラッシュされていることを確認し、イソフルランをオフにして、フラッシュが始まるとノーズコーンを外してください。次に、上記で詳述カテーテル（3.2）と、さらに5分間圧力灌流にホルマリンで満たされたシリンジを取り付けます。最後に、コントラストと、最後の注射器を灌流ゆっくり5分間マウスに剤。 </li><li>オプション：写真カメラで血管系にMicrofilだけ動脈構造が灌流されていることを確認する。 </li><li> 4で10％NBFや店舗°CO /造影剤重合用Nを充填した容器内にマウスを置きます。 24時間後、10％NBFをリフレッシュし、解剖ツールを使用して胴体の下半分（下腹部大動脈結紮に対して遠位）で皮膚を除去し、室温で4日以上のために容器にマウスを後ろに置きます。 </li><li>対側と虚血性後肢を解剖し、骨の脱灰を促進するために48時間ギ酸溶液（カル - 例II）に標本を置きます。その後、1時間と10％NBFへの復帰のために流水で検体をすすぎ、Faxitron（骨が脱灰されていることを確認するために使用されるX線システム）およびマイクロCTイメージングを進める。 </li></ol>

<ol>
	<li>Epstein, S. E., Fuchs, S., Zhou, Y. F., Baffour, R., Kornowski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+interventions+for+enhancing+collateral+development+by+administration+of+growth+factors:+basic+principles,+early+results+and+potential+hazards.">Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards.</a> Cardiovasc. Res. 49, 532-542 (2001).</li><li>Esaki, J., Marui, A., Tabata, Y., Komeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+systems+of+angiogenic+growth+factors+for+cardiovascular+diseases.">Controlled release systems of angiogenic growth factors for cardiovascular diseases.</a> Expert Opin. Drug Deliv. 4, 635-649 (2007).</li><li>Bentley, M. D., Ortiz, M. C., Ritman, E. L., Romero, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+microcomputed+tomography+to+study+microvasculature+in+small+rodents.">The use of microcomputed tomography to study microvasculature in small rodents.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, 1267-1279 (2002).</li><li>Golomb, B. A., Dang, T. T., Criqui, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+arterial+disease:+morbidity+and+mortality+implications.">Peripheral arterial disease: morbidity and mortality implications.</a> Circulation. 114, 688-699 (2006).</li><li>Hiatt, W. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACC/AHA+Guidelines+for+the+Management+of+Patients+with+Peripheral+Arterial+Disease+(lower+extremity,+renal,+mesenteric,+and+abdominal+aortic):+a+collaborative+report+from+the+American+Associations+for+Vascular+Surgery/Society+for+Vascular+Surgery,+Society+for+Cardiovascular+Angiography+and+Interventions,+Society+for+Vascular+Medicine+and+Biology,+Society+of+Interventional+Radiology,+and+the+ACC/AHA+Task+Force+on+Practice+Guidelines+(writing+committee+to+develop+guidelines+for+the+management+of+patients+with+peripheral+arterial+disease)--summary+of+recommendations.">ACC/AHA Guidelines for the Management of Patients with Peripheral Arterial Disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Associations for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (writing committee to develop guidelines for the management of patients with peripheral arterial disease)--summary of recommendations.</a> J. 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著者らは、利害の対立を宣言しません。全ての動物の手順は、実験動物のケアと使用に関するガイドラインに従って実施され、ヒューストンのテキサス健康科学センターの大学制度アニマルケアと使用委員会（IACUC）によって承認された。

ここでは、後肢虚血のマウスモデルにおける浸透薬物/物質の送達のための方法を提示する。また、我々は血管網の分析のための3次元再構成を生成するためにMicrofilを使用している鋳造技術を説明する。 虚血の程度または重篤度は、動脈結紮/切除がどこで行われるかに応じて異なります。我々は大腿深とepigastrica動脈の起源の近位大腿動脈の二重結紮を作成しましたが、?...

末梢動脈疾患（PAD）は、脚部4に不十分な血液供給を引き起こすアテローム性動脈硬化症である。それは8から12000000までのアメリカ人に影響を与え、現在の治療法は限られた救済5,6を提供しています。足の血液循環を改善する新規な治療剤は疾患の進行を抑制するだけでなく、生活の質を向上させないだろう。しばしば高齢の患者で見られる減少腎臓や肝臓の機能が薬物代謝を減少させ、全身投与で副作用を増やすことができますので、PADの発生率は50歳以上の人々に高くなっているので、地元の薬理学的療法は、より望ましい治療法である。 したがって、我々は、局所的に投与薬剤が血管新生および血管リモデリングを促進することにより、後肢虚血を軽減するかどうかを調べるために、PADのマウスモデルを作成しました。具体的には、連続的に治療薬を送達するためにカテーテルを挿入浸透圧ポンプを用いマウスの虚血性大腿筋。我々の送達システムを用いて、ローカル環境での薬物の最適な濃度を維持することができたが、このアプローチは、適切な薬剤の生物活性を可能にする可能性全身性副作用を回避する、および全身投与に関連する限られた局所薬物アクセスの欠点を克服する。また、ローカルで管理エージェントは血行再建を促進するかどうかを評価するために、我々は、高度な鋳造と微小血管の変化の定量化を可能にする高精細イメージング技術を使用していました。総称して、このビデオ記事で使用方法の組み合わせは、PAD患者の7-9に薬理学的に誘導された血管再生の理解を助けるための前臨床試験で有用である。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>Surgical tools</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Puritan sterile cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>22-029-499</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Betadine (povidone-iodine)</td>
			<td>Fisher Scientific</td>
			<td>19-065534</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>70% Alcohol pads</td>
			<td>Fisher Scientific</td>
			<td>NC9926371</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>6-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>8709</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>8-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>2775</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Osmotic pump</td>
			<td>ALZET</td>
			<td>1002</td>
			<td>Type: Tool, 14 day release</td>
		 </tr>
		 <tr>
			<td>Vinyl catheter</td>
			<td>ALZET</td>
			<td>7760</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Heparinized saline (0.9%)</td>
			<td>Baxter</td>
			<td>2B0944</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Neutral buffered formalin</td>
			<td>Richard-Allan Scientific</td>
			<td>5705</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Microfil (silicone rubber contrast agent)</td>
			<td>Flowtech</td>
			<td>MV-112</td>
			<td>Type Reagent, Microfil White</td>
		 </tr>
		 <tr>
			<td>Cal-Ex II (formic acid solution)</td>
			<td>Fisher Scientific</td>
			<td>CS511-1D</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Buprenex</td>
			<td>CIII</td>
			<td>7571</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td>Bupivicaine</td>
			<td>Hospira, Inc.</td>
			<td>381</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Dissecting microscope</td>
			<td>Carl Zeiss Microimaging</td>
			<td>Zeiss Stemi 2000-C</td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Laser Doppler perfusion imager</td>
			<td>Perimed Inc.</td>
			<td>Periscan PIM3 </td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Micro-CT imaging system</td>
			<td>GE Healthcare</td>
			<td>Explore Locus SP </td>
			<td>Type:Equipment</td>
		 </tr>
		</tbody>
 </table>

osmotic drug delivery to ischemic hindlimbs and perfusion of vasculature with microfil for micro-computed tomography imaging

末梢動脈疾患の動物モデルにおける前臨床研究では、微小循環を刺激するように設計された治療薬の有効性をテストする上で重要な役割を果たしている。投与経路が深く、これらの薬剤1,2の生物活性と有効性に影響を与えるため、これらの薬剤のための配送方法の選択は重要です。本稿では、局所的にカテーテルを挿入浸透圧ポンプを用いて、虚血性後肢中の物質を管理する方法を示します。このポンプは、時間の割り当てられた期間に連続して水溶液の一定量を供給することができます。また、左後肢に大腿深とepigastrica動脈の起源の近位大腿動脈の結紮により誘発される一方的な後肢虚血の我々のマウスモデルを提示する。最後に、我々はMicrofil、シリコーン放射線不透過性キャスティングエージェントと大動脈腹部大動脈と後肢の血管系の灌流のin vivoカニュレーションとライゲーションで説明。 Microfilは灌流と塗りつぶし全体の血管床（動脈と静脈）を、我々は出口のための主要な血管の導管をライゲーションしているため、エージェントは小標本のマイクロCT 3を利用した将来のex vivoでのイメージングのための血管系内に保持することができますことができます。

アニメーションは、浸透圧ポンプの挿入とプロトコルに詳述後肢虚血手術を示しています。 図1は、虚血を確認し、後肢の灌流のレーザードップラー画像を示す。 Microfilと血管鋳造後、血管網の3次元マイクロCT画像がMicrofil効果的に血管を埋めることができることを示している（ 図2A）が、その不連続性が発生する可能性があります（ 図2B）により、様々...

Watch this Scientific Journal Video about Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging at JoVE.com

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

我々はここに表示<em生体内で&gt;</em一定の局所薬物送達およびマウスモデルにおける後肢虚血を作成するための浸透圧ポンプの&gt;挿入。また、後肢の脈管構造は、マイクロコンピュータ断層撮影（マイクロCT）イメージングのために準備するために、Microfil、シリコーン放射線不透過性物質で灌流されている。

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 ...

osmotic-drug-delivery-to-ischemic-hindlimbs-perfusion-vasculature

Research

JoVE Journal

Medicine

12.2K ビュー.  The Texas Heart Institute at St. Luke's Episcopal Hospital. 我々はここに表示挿入。また、後肢の脈管構造は、マイクロコンピュータ断層撮影（マイクロCT）イメージングのために準備するために、Microfil、シリコーン放射線不透過性物質で灌流されている。

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

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

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

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 ...

医学

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 ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

マウスの動静脈瘻内皮への管腔内薬物送達

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

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 ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

ラット肝臓肝門部クランプモデルを用いたダイレクトセグメント別肝臓内送達方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Ivacaftor、主要な代謝産物および嚢胞性線維症患者の体液中の Lumacaftor の臨床サンプルの高スループット分析のための最適化された LC-MS/MS 法

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

体外血液脳関門ブタ脳血管内皮細胞に基づくモデルの確立のための改良法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

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.