この作品は、日本学術振興会AEHのための科研費（グラント番号25461196）およびTB（グラント番号23390218と15H04833）からのイン援助グラント-と国立衛生研究所HL111190（DMO）を付与することによってサポートされていました。著者らは、動物の遺伝子型決定および組織切片の準備を手伝うこと​​に彼女の努力のためのみゆき山本を承認したいと思います。我々は、技術サポートおよび試薬のための共同研究資源、医学部、慶應義塾大学に感謝しています。

動物実験は、慶應義塾大学の施設内動物管理使用委員会によって承認されました。 注：本研究では、Sftpc-のrtTA及び肺腺癌が急速にドキシサイクリン6,7を含む飼料の供給により誘導後開発したトレ- FGF9-IRES-EGFP DTマウスを使用していました。しかし、すべての評価手順は、他の肺癌マウスモデルに適用することができます。 1.実験概要： <ol><li>ベースラインでの肺の状態を特定します。 <ol><li>腫瘍誘発する前に、DTマウスは8である場合 - 12週齢、第一のマイクロCTスキャンを（下のセクション2と3を参照）を行います。これは、肺のベースラインスキャンとして機能リーキー導入遺伝子によって引き起こされる自然発生的に開発した結節がないことを確認し、腫瘍誘導前、既存の肺病変が存在しないことを文書化します。 </li></ol></li><li>腫瘍誘導を開始します。 <ol><li>正規のCHからDTマウスを切り替えますドキシサイクリン飼料にOW肺胞細胞に線維芽細胞成長因子（FGF）9発現を誘導し、腫瘍成長を開始します。ドキシサイクリン飼料（200 ppm）で自由摂取を与えます。 </li></ol></li><li>マウス肺における腫瘍小結節の発達を確認します。 <ol><li>誘導前のスキャンと比較して腫瘍結節の開発を識別するためのマイクロCTスキャンを実行します。 </li></ol></li><li>治療に対する反応を評価します： <ol><li> 、治療に応答して腫瘍結節の変化を検出FGF受容体（FGFR）阻害剤AZD4547を管理するためのマイクロCTの能力を試験するために、その後、5および10週間後に追加のマイクロCTスキャンを実行します。 </li></ol></li><li>エンドポイントの評価： <ol><li> （下記のセクション6を参照）、組織学的評価のために、すべての治療群と対照マウスとプロセス組織を安楽死させます。 </li></ol></li></ol> 2.マイクロCT画像取得のためにマウスを準備します。 <ol><li>マイクロCTスキャナとコンピュータの電源を入れます。 </li><li> clicクリック「R_M CT2」という名前のソフトウェア上でkは、その後、「ウォームアップ」をクリックしてください。 </li><li>マウスは、マイクロCT室から置かれる時に、サンプル台を取り外します。ラップで床を包みます。 </li><li>顕著なレベルまで麻酔気化器にイソフルランを追加することで、麻酔導入ボックスを設定します。 3L /分でイソフルラン流量を設定します。 </li><li>インダクションボックスへの酸素の流れを開始し、1L /分の流量を設定するために酸素タンクを開きます。 </li><li>麻酔導入ボックスにマウスを置いて、それが深く、自発運動の欠如によって、および（組織損傷や皮膚のブレークが発生しない皮膚の小さな倍の穏やかなピンチ、）皮膚のピンチに応答して麻酔をかけていることを確認します。 </li><li>麻酔中に角膜の乾燥を防ぐために、眼の潤滑剤を塗布します。 </li><li>マイクロCT室を開き、サンプルのベッドの上で背側を上にしてマウスを置きます。マウスの頭部を持ち、オードにおける下肢から下方にボディを引っ張りますrはストレッチと対称的に体をまっすぐにします。 </li><li>インダクションボックスに麻酔の流れをオフにして、マイクロCT室に接続する管に向かってそれをオンにします。 </li><li>連続麻酔薬を投与するために、マウスの鼻の中に麻酔チューブを置きます。 </li><li>位置にマウスを固定するために、マウスやプラスチックラップでサンプルのベッドを包みます。 注：深い麻酔下で、スキャン中の任意のわずかな動きや、その本体のねじれがかすんイメージと解釈することが困難になりますので、サンプル床に包まれたマウスを維持することが重要です。 </li><li>マイクロCT室を閉じます。 </li><li> 90 kVの160μAと4.5分のスキャン時間にマイクロCTシステムを調整します。 50×50×50μmである24×19ミリメートルとボクセルサイズの画像範囲を設定します。ハートビートのための同期モードを使用してください。 </li><li>そのフォルダに新しい画像を保存するために、「データベース」に新​​しいフォルダを作成します。 </li><li>スキャンを開始します。 </li><li>スキャンが完了した後、空のケージにマウスを移動し、それが意識を取り戻すまでそれを観察します。それは完全に麻酔の影響から回復するまで、他のマウスとそれを入れないでください。 </li></ol> 3.事前誘導マイクロCT画像の可視化と分析： <ol><li> ：マイクロCT画像を可視化するために、次のWebサイトから無料ImageJソフトウェアをダウンロードし<a href="http://imagej.nih.gov/ij/">http://imagej.nih.gov/ij/</a> 。注：すべてのマイクロCTデータファイル約500 TIFファイル（.tifファイル）のスタックです。他の画像閲覧ソフトウェアを使用することもできます。 </li><li>シリアルマイクロCT画像ファイルを開き、その逆の腹部またはに首から各マウスのすべての画像をスクロールします。 </li><li>ナイーブ野生型マウスのスキャンを使用して、マウスの解剖学の知識に基づいて、胸部内の ​​異なる領域の密度は、通常の解剖学的構造を特定する（ 図1A参照 ）。 <ol><li>でカーソルを保持しますコンピュータのマウスと白っぽい腹部内臓や横隔膜から、胸を通って首まで開始、スクロールアップ。 </li><li>骨の胸のケージのランドマーク（両側に背中と肋骨で正面に胸骨、椎骨）を特定します。 </li><li>胸の前、心臓に近いと縦隔の主要な血管に心を識別します。 </li><li>その後、より小さな気管支に分岐していき、左右の主気管支に分岐する、（首と胸の上部のレベルでの小さな暗い円として）気管内腔を観察します。各気管支が密接に2または3の血管（ 図1A）に関連付けられていることに注意してください。 </li></ol></li><li>非誘導DTマウスのスキャンを調べる起動し、異常の有無を識別します。 <ol><li>任意のさらなる実験から異常な誘導前の肺の影（ 例えば、結節、気腫性嚢胞、 等 ）を有するマウスを除外します。 （ 図1C、E、G、H </strong&gt;）。 </li></ol></li></ol> 4.腫瘍の誘導： <ol><li>正常な肺のスキャンを示した実験マウスにおける腫瘍発生を誘導するために、ドキシサイクリン含有飼料（200 ppm）でに、通常の固形飼料から自分の食べ物を切り替えます。 </li></ol> 5.フォローアップスキャン： <ol><li> 10週間後、それらの肺における腫瘍小結節の発達を確認するために、すべてのマウスの第2のマイクロCTスキャンを実行する（ 図2参照 ）。 </li><li>二つのグループにマウスを分割します。一つのグループにし、10週間以上のために他の対照群とプラセボ（10週間/週6日間、胃管を経由して125μgの/ kg /日）FGFRブロッカーAZD4547を管理します。 </li><li> 5週間後 - 4第3スキャンを実行することにより、結節性陰影の変化をフォローアップ。 </li><li>治療の10週の終わりには、第4の走査を行います。 </li><li> CO 2吸入または0.1ミリグラム/ペントバルビタールを200μlの腹腔内注射で全てのマウスを安楽死させます。 </li><li>にその後、任意の異常陰影の出現/消滅（ 図3をチェック二つ以上の異なる時点で同じマウスのシリアルスキャン画像内の同様の位置を特定し、誘導後および処理に応答した腫瘍結節の動的変化を識別）。 </li><li>異なる時点で、同じマウスにおける同一平面上の識別を容易にするために、マウスの胸部内の解剖学的ランドマーク構造に興味のある面を関連付けるしてみてください。 <ol><li>このような気管、その分岐部、左右の主気管支、大動脈、横隔膜、および大血管などのランドマークを使用してください。 注：胸椎、肋骨、胸骨を含む胸部の骨理由は、スキャン位置の誤った解釈の可能性を高めるサンプルベッドの上でマウス本体の位置合わせで共通のマイナー傾く、の位置決め目印としてあまり有用です。同様に、スキャンファイル内の画像のシリアル番号がidentifyinために信頼性がありませんグラム経時ため、別の時点からマウス本体の長さの変化の同じ位置。異なる時点で同一平面を特定するのに失敗した場合や不正確さが所見の偽陽性/陰性の解釈になることがあります。 </li></ol></li></ol> 6.マウスの安楽死と肺コレクション： <ol><li> CO 2吸入または0.1ミリグラム/ペントバルビタールを200μlの腹腔内注射でマウスを安楽死させます。 </li><li>腹壁を通って長手方向に切断することによって腹部の内臓を公開します。腹部大動脈を切開することにより、肺の中の血液の量を減らすために、マウスをブリード。 </li><li>細かいハサミで振動板をスリット。これにより、肺の崩壊、胸腔からの負圧の損失をもたらします。前胸壁のリブの部分を切断して除去することにより、肺と心臓を露出。 tracheを公開するために、皮膚および軟組織を切断することによって、首の前面部分を清掃してくださいA。 </li><li>心臓や胸腺を切り取ります。食道からそれを分離するために気管の後ろに鉗子を挿入します。 </li><li>その後、G24カニューレと気管にカニューレを挿入、挿入部分の周りに糸を締めて所定の位置に固定します。 </li><li> 25センチメートルカラムを用いて気管カニューレを介して氷冷4％パラホルムアルデヒド（PFA）を使用して肺を膨らませると固定します。カニューレを外し、喉頭への結合から、上部気管を切断し、その後、PFAの漏洩を防止するためのスレッドを締めます。 </li><li>縫合糸から気管を引いて、その添付ファイルから、それを分析し、 -ブロックエン肺でそれを削除するには、下方向に続けています。 4％PFAの5ミリリットルを含む15mlチューブに挿入します。標準のパラフィンブロック8に組織を処理した後、完全な組織浸透、定着を確実にするために、PFA、O / Nで肺を残します。 </li><li>標準的な技術を用いてヘマトキシリンおよびエオシンでミクロトームや汚れに6μmの厚さにスライスし、パラフィンブロックをカット。 </li></オール&gt; 7.組織学的評価： 注：デジタル組織学的評価のために、「スライドスキャナ」の使用は、ここに記載されているが、評価のための定期的な顕微鏡および視覚組織学的評価を使用することも可能です。 <ol><li>スライドスキャナ装置とコンピュータの電源を入れます。 </li><li> 「NDPスキャン」ソフトウェアをクリックします。 </li><li>スライドのシリーズをスキャンするためのスキャンモード」のスライドのバッチ」と「半自動モード」を選択します。 注：順次走査中にスライドを取り出し、ロボットアームは、スライドガラスの縁部上の凹凸に非常に敏感です。 </li><li>マシンにそれらをロードする前に、すべてのガラススライドのエッジを触診します。カバーガラスまたは乾燥した封入剤のいずれかの突起がある場合は、カッターやメスでそれを清掃してください。 </li><li>スライドカセットにスライドをロードします。試料のハッチを開き、所定の位置にカセットをスライドさせて「1」;。ドアを閉める。 </li><li>コンピュータ・ソフトウェアでは、「OK」ボタンをクリックして、カセットの対応する位置にあるすべてのスライドに短い説明的な名前を付けます。 </li><li> 「明」：プロファイルモードを選択します。 </li><li>暫定スキャンを開始するには、「バッチを開始」をクリックします。 注：マシンはすべてのスライドをスキャン終了すると、ソフトウェアは自動的にすべてのスライド上の組織と領域を検出し、関心領域としてそれを提案します。 </li><li>必要な場合は、マウスの左ボタンを押しながら、地域の境界線を引くことによって、関心領域を再定義します。 注：関心領域としてスライドの過大な領域を定義することははるかに長いス​​キャン時間になります。 </li><li>すべてのスライド上の利益を地域に満足したら、すべてのスライドのスキャンを開始するには、「スキャン」をクリックしてください。 注：スキャンしたファイルは、低解像度と高解像度でデジタル的に観察することができ、画像はJPEGとしてエクスポートすることができますファイル。 </li></ol>

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The authors declare that they have no competing financial interests.

腫瘍結節の開発や、より計画する肺癌関連の実験を行っている科学者を可能にする肺癌動物モデルにおける治療に対する反応のリアルタイムの肺の異常の識別と監視のためにここで説明するマイクロCTに基づく方法正確かつ効率的な実験は、時間とリソースを節約しながら。我々は以前、同じ目的の6のためにMRIを使用しています。 MRIでの肺結節の検出のためのスキャンおよびしきい?...

肺癌は世界1の周りの癌死の主要な原因です。肺がんの予防、早期発見、および治療 ​​の研究は世界2,3の多くの研究センターで進行中です。肺癌のためのいくつかの動物モデルが開発されており、それらは、起源の肺発癌および細胞のメカニズムの研究において、癌幹細胞の存在を決定する際に、種々の新規な治療戦略4を検査するのに有用であることが証明されています。以前のモデルは、マウス5の感受性株に発癌物質誘発性腫瘍の開始に依存していました。具体的に操作遺伝子損傷の結果としてのノックアウトと肺がんが発生したトランスジェニックマウスモデルの開発は、実質的に腫瘍の誘導を制御し、ヒト肺癌4のいくつかの側面を模倣する能力を向上させました。しかしながら、肺癌の動物モデルの使用における主要な課題は、リアルタイムの方法が存在しないことです正確に識別して、マウスの肺の腫瘍の発症や進展を監視し、そのような治療に応答して、それらの継続的な成長または減少としてそれらのサイズ、で、それ以降の変更を文書化します。これは、腫瘍を同定するために、それらの実験の結果を評価するために、いくつかの時間、労力、およびリソースを消費する技術に頼ら研究を余儀なくされました。腫瘍の誘導に応答する固有の間マウスの変動の存在は、データのばらつきを低減するために、各実験群の動物の多数の使用を必要とします。リアルタイムで処理し、腫瘍の成長または応答を評価することができないことは、サンプルからの資源の浪費で、その結果、盲目的に彼らが正しいデータを収集することを保証するために長時間の実験プロトコルで複数の時点でマウスを安楽死させるために、研究者を余儀なくされました早すぎたり遅すぎたりしている時点で収集。 本研究では、この方法は、小動物マイクロ-Cを利用しますomputed断層撮影（マイクロCT）マウスを生きているに肺腫瘍を検出し、フォローアップするためのスキャナーが導入されています。私たちは、最近記載Sftpc-rtTAのとトレ- FGF9-IRES-EGFPのダブルトランスジェニック（DT）急速にドキシサイクリン6,7-による誘導後の肺腺癌を発症するマウスを使用しました。マイクロCTの使用は、（特に）に私たちを可能に誘導する前に、異常な肺の異常でマウスを除外し、誘導後肺の腫瘍結節の発達を確認し、実験的治療に反応して腫瘍結節の変化を観察します。エンドポイントマウスの安楽死および組織学的評価は、マイクロCTを用いて行っリアルタイム評価の精度を確認しました。私たちは、この技術は、貴重な資源を節約観測時間を短縮し、結果の正確さと理解を向上させながら肺癌動物モデルを用いて、より良い計画実験を行うための道を開くだろうと信じています。

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			<td height="21" style="height:21px;width:319px;">micro-X-ray&#8211;computed tomography</td>
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			<td height="21" style="height:21px;width:319px;">Isoflurane Vaporizer - Funnel-Fill</td>
			<td style="width:264px;">VETEQUIP</td>
			<td>911103</td>
			<td></td>
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			<td height="21" style="height:21px;width:319px;">Induction chamber, 2 Liter&#160; W9.5&#215;D23&#215;H9.5</td>
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			<td style="width:140px;">941444</td>
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			<td height="21" style="height:21px;width:319px;">Isoflurane</td>
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			<td>A-1088</td>
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			<td height="21" style="height:21px;">Pentobarbital</td>
			<td style="width:264px;">Kyoritsu</td>
			<td style="width:140px;">SOM02-YA1312</td>
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			<td height="21" style="height:21px;">G24 cannula&#160;</td>
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			<td style="width:140px;">SP-FS2419</td>
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			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td style="width:264px;">Wako</td>
			<td style="width:140px;">163-20145</td>
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			<td height="21" style="height:21px;width:319px;">Microtome</td>
			<td style="width:264px;">Leica</td>
			<td style="width:140px;">RM2265</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Doxycycline</td>
			<td style="width:264px;">SLC Japan/PMI Nutrition International</td>
			<td style="width:140px;">5TP7</td>
			<td></td>
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			<td height="21" style="height:21px;">ImageJ software&#160;</td>
			<td style="width:264px;">National Institute of health</td>
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			<td>http://imagej.nih.gov/ij/</td>
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			<td height="21" style="height:21px;width:319px;">Puralube vet ointment (Occular lubricant)</td>
			<td style="width:264px;">Dechra</td>
			<td style="width:140px;">NDC 17033-211-38</td>
			<td></td>
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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.

肺の異常を有するマウスの同定は、ベースラインで行いました。 DTマウスは8であった腫瘍誘導、前 - 生後12週間、すべてのマウスの肺は、マイクロCTを用いてスキャンしました。驚くべきことに、マウスの約50％は、その後の研究に含めるためには不適切と判断する私たちを強制的に異常を示しました。これらの異常は、結節状陰影、大規模な単一または複数の小さな...

Watch this Scientific Journal Video about Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer at JoVE.com

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

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

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.

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

using-micro-computed-tomography-for-assessment-tumor-development

Research

JoVE Journal

Medicine

10.6K ビュー.  Keio University School of Medicine. 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.

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

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

手術標本の腫瘍外植片を用いて新規抗がん薬の開発のための方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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 ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

弱視における抑制の測定と治療

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

同所性膀胱腫瘍モデルと膀胱内Sarnaの治療の評価

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

ゼブラ土着腫瘍モデルを用いたメラノーマの修飾のためのスクリーニング

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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 ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

エキソビボ処理前臨床のための原発腫瘍および/ ​​または関連した転移の応答と治療薬の臨床開発

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

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 ...