この研究は、アリゾナ州にJDLとNIH / NCIの助成金＃CA120711 - 01A1とCA120711 - 01A1にCCSRIグラント＃700537とCIHRグラント＃84535によってサポートされていました。全ての実験は、動物実験の規制やガイドラインに従って実施し、ウェスタンオンタリオ大学で、カリフォルニア大学サンディ​​エゴ校の委員会、動物実験を使用していた。

1。鳥類胚のCAMへの腫瘍の接種<ol><li> 8のようなシェルレス受精ニワトリ胚を準備します。 </li><li>胚発生の9日目に、1 mLシリンジに接続して18ゲージ針を用いて、マイクロインジェクターを組み立てます。タイゴンチューブの5〜6インチピース（1 / 32&quot;内径、3 / 32&quot;外径、1 / 32&quot;壁厚）をカットし、慎重にチューブに針のベベルを挿入する。チューブの約4〜5インチのはず針の先端（図1a）から延びる。 </li><li>細胞懸濁液と注射器とチューブを埋める。その後、チューブの端にマイクロインジェクションのガラス針を挿入し、慎重に気泡を取り除く。 </li><li> CAM（図1c）内ボーラスとして10,000-100,000癌細胞とイルミで解剖の範囲で9日目胚（図1b）を注入する。徐々に細胞を注入し、慎重に針がCAM内に表示されるボーラスを形成する細胞の正しい場所にあることを確認してください。 CAMの表面にドリップをキムワイプまたは他のアプリケーターを用いて洗浄することができる細胞。 </li><li> 60％の湿度で38℃で加湿インキュベーターに胚を戻すと成長し、血管が発達する（最大7日間）に腫瘍ができます。 </li></ol> 2。ナノ粒子の調製<ol><li>ニワトリ胚への注入のために蛍光標識CPMVのナノ粒子を準備するには、。いずれかを削除する1分間のために使用し、遠心前にウイルス粒子を（100μg/ mlの濃度に、PBS、pH7.4中8のように合成された渦混合物も希釈凝集体。音波処理も複合体および凝集の程度に応じて役に立つかもしれません。4℃で保存した場合、蛍光標識CP​​MVsの株式は、少なくとも1年間は安定である。·ダークのC。夫婦を入れて定期的に株式をチェックして、スライドガラスの上に落とし、蛍光。透過型電子顕微鏡（TEM）をチェックすると、粒子が抱合した後、そのまま残っているかどうかを判断するために使用することができます。 </li><li>簡単に言うと、ウイルス粒子の1マイクログラムは、（2μlの最終容量で）銅被覆グリッドに追加することができます。次に、電子顕微鏡（フィリップスEM 410）で1分間、2％酢酸ウラニルの等量を追加します。 </li></ol> 3。蛍光標識ウイルス粒子の静脈注射<ol><li> 16日目に、上記のように、マイクロインジェクターを組み立てます。注射器にチューブ（&gt; 200μL）を介してウイルス粒子の所望の体積を描く。慎重に気泡を取り除く。チューブの端にマイクロインジェクション用ガラス針を挿入します。針が（図2a）できるだけ長くしてテーパー状になっていることを確認してください。針が非常に鈍い場合は、外胚葉を通して貫くことができなくなりますし、血管を貫通して失敗します。針が非常にシャープの場合は組織を貫通する際に、先端が収縮されます。 </li><li>静脈内（図3D）可視化する目的のサイトから遠位の腫瘍を有する胚に1 mg / mlのフルオレセインデキストラン（MW7万ダ、Invitrogen社）の100μlを注入する。 CAMの静脈の成功カニューレは、インジェクションフローのパス内の血液のクリア（図2b）によって明らかである。注入後の血管の漏れ1時間を評価する。 </li><li>静脈内に目的のサイトから遠位血管の漏れで腫瘍を含む胚には1mg / CPMV -のAlexa Fluor ® 647（CPMV - AF 647）やPEG化CPMV -のAlexa Fluor ® 647（CPMV - PEG - AF 647）mlの溶液50μlを注入する可視化すること。 </li><li>画像の即座に腫瘍と横河電機回転ディスク、浜松ImagEM 9100〜12 EM - CCDカメラを装着したツァイス審査Z1正立顕微鏡を用いて注射後時間ごと。 </li></ol> 4。リアルタイム生体イメージング<ol><li>胚の撮像ユニットを組み立てるには、胚のイメージングユニットの蓋の下側の画像ポートの周囲に真空グリスの薄層を適用し、ポートに18 mmのガラスカバーを装着します。 </li><li>カバースリップは、イメージングのための必要なエリアをカバーするような胚を配置します。ゆっくりとカバースリップがちょうど胚に接触するまでふたを少し下げて、その後（図2c）の場所でそれを保持するためにユニットの上に蓋をねじ込む。 </li><li> ° C胚を含む皿の外胚の撮像ユニットに37に加熱水を追加し、その後、37に平衡内部環境室℃で直立共焦点顕微鏡のステージ上に全体のユニットを配置</li><li>スピニングディスク共焦点蛍光顕微鏡（図2E）の下で胚を含む撮像ユニットを置きます。撮像ユニットは、キャプチャされる三次元Zスタックやタイムラプスイメージの両方を可能にする、場所に胚を保持して画像を取り込む際に固定されたビューのフィールドを保持します。我々は、パーキンエルマーの（旧即興）Volocityのソフトウェアパッケージを（図3a）を用いて3次元タイムラプス画像を取得し、分析する。腫瘍の三次元スタックの高解像度とdetaile可視化するために周囲の血管系を取得する特定の時間点でのD構造解析。腫瘍血管系の詳細な構造変化をマッピングするために定期的な時間の点で三次元スタックを取得する。画像の腫瘍注射後の時間ごと。 </li><li>腫瘍や間質内の選択した領域内の平均値のAlexa Fluor（AF）647信号（非腫瘍領域）Volocity（パーキンエルマー）等の画像の定量ソフトウェアを用いて計算することにより、ウイルス粒子の取り込みを定量する。間質比は腫瘍は間質の平均AF 647信号を介して腫瘍の平均AF 647信号を割ることにより算出した。 1より高い腫瘍/間質比は、ナノ粒子が腫瘍によって占有されていることを示します。 </li></ol> 5。代表的な結果： この例ではここで説明する、私たちは9日目ニワトリ胚（図1b）のCAM内に大きさで1mm程度ボーラスを形成するために、HT - 29結腸癌細胞を注入した。接種後、胚は十分な腫瘍の増殖と血管新生（図1c）を許可する加湿インキュベーターで7日間培養した。胚は、腫瘍の血管新生を確認するために、低分子デキストランを静脈内注射した、と腫瘍はツァイスAxioExaminer Z1正立顕微鏡（図2d）で可視化した。 CPMV - AF 647またはCPMV - PEG - AF 647ナノ粒子（図3aとb）の静脈内投与後、高解像度のリアルタイム共焦点イメージング（図2eは）CPMVとCPMV - PEGは、ナノ粒子の両方が急激に全体の血管系のラベルが付いたことを明らかにしたが、腫瘍によるCPMV - PEGの取り込みは、12時間（図3a）の後CPMVより約3倍高かった。ナノ粒子の相対的な腫瘍の取り込み、画像解析ソフトウェアを（パーキンエルマーからVolocity）を用いて決定した。関心領域は、腫瘍内と外で選ばれた（間質コンパートメントにおける）とそれぞれの平均蛍光強度を測定した。データは、腫瘍/間質比として表現さ​​れます。 <img src="/files/ftp_upload/2808/2808fig1.jpg" alt="図1"/> 図1。鳥類の胚のCAMへの腫瘍のマイクロインジェクション。示されているように（a）にマイクロインジェクション装​​置は、コンポーネントから組み立てられます。 （B）9日目での鳥の胚では、CAMが表面全体を覆​​うように広がっているときに腫瘍を接種する準備が整いました。 （C）16日目では、腫瘍は直径1cm（破線）にまでに成長し、ナノ粒子を注射するための準備ができているでしょう。 <img src="/files/ftp_upload/2808/2808fig2.jpg" alt="図2"/> 図2ナノ粒子の注入と生体内イメージング。 CAMの静脈に注入するインジェクターの針の先端は準備ができて（）を示すと（b）マイクロインジェクターの針が静脈に挿入した（矢印）とクリアで見られる血流（に注入ナノ粒子解剖顕微鏡下で注入血液の）。 CAMとの直接インタフェースカバーと鳥類の胚を含む（C）イメージングユニット。 （D）ナノ粒子の注入に先立って、腫瘍の血管新生は、フルオレセインデキストランの投与後に生体内イメージングを用いて評価する。 （e）のイメージングユニットは37℃に温度調節エンクロージャのセット内で直立共焦点顕微鏡のステージ上に位置する胚を含む<img src="/files/ftp_upload/2808/2808fig3.jpg" alt="図3"/> 図3。ヒト腫瘍におけるナノ粒子の取り込みの生体内可視化。腫瘍は、（a）CPMV - AF647と（b）CPMV - PEG - AF647の注入後7時間を可視化している。その後の分析のための鳥類の胚から腫瘍のd）の切除。

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利害の衝突は宣言されません。

鳥類の胚の漿尿膜（CAM）は、ヒトの腫瘍の血管動態と薬物動態を評価するための有用なモデルです。 CAMモデルの構造と位置は、高品質の画像取得を可能にし、侵襲的な外科手術なしで癌異種移植片の多くの種類の対応。また、がんの腫瘍異種移植片は、腫瘍組織におけるナノ粒子の蓄積を評価するために、迅速、安価でセミハイスループットな手段を提供し、7日以内に血管が新生になる漿尿膜に移植。シェルレスニワトリ胚のCAMに移植された癌の異種移植片は、腫瘍血管系におけるナノ粒子の取り込みに関する直立落射蛍光または共焦点顕微鏡、文脈と時間情報の高分解能光学系にアクセスすることができるので容易に得ることができる。このモデルにおける癌の異種移植片は深さ未満が200メートルを維持しながら、大きい腫瘍で、その結果、CAMを介して横方向に成長する傾向がある。標準の落射蛍光顕微鏡は、効果的に腫瘍全体の質量を貫通することができるので、これは彼らが特に生体内イメージングに適しています。対照的に、マウスの中で表在性または同所のサイトのいずれかで移植された腫瘍は、それは難しい正確に非侵襲的手法によりこれらの腫瘍内の深いナノ粒子をローカライズすること、三次元的に増殖する。我々は、ナノ粒子製剤の広い範囲ののin vivo解析に適していることがこのモデルの可能性を強調し、ヒト腫瘍異種移植片の数で量子ドット、リポソーム、および酸化鉄ナノ粒子の取り込みを評価するためにこのモデルを利用してきた。

<table border="1"><tbody><tr><th>試薬名/機器</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>受精レグホン種の卵</td><td>フレイの`sの孵化場、セントジェイコブス</td><td> N / </td><td></td></tr><tr><td>ドレメルロータリーツール</td><td>ドレメル</td><td></td><td>任意のモデルを使用できます。 </td></tr><tr><td>ドレメルのカットオフホイールありません。 36 </td><td>ドレメル</td><td> 409 </td><td></td></tr><tr><td>スポーツマンハッチャー</td><td>ベリーヒル</td><td> 1550HA </td><td></td></tr><tr><td>スポーツマンインキュベーター</td><td>ベリーヒル</td><td> 1502EA </td><td></td></tr><tr><td>エタノール</td><td></td><td></td><td> 70％（vol / vol）の</td></tr><tr><td>ポリスチレンは、ボートの重量を量る</td><td> VWR </td><td> 12577〜01 </td><td></td></tr><tr><td>正方形のペトリ皿</td><td> Simport、VWR </td><td> 25378-115 </td><td></td></tr><tr><td>ふたが付いているラバーメイドラバーコンテナ</td><td> Guillevin </td><td> RH3 - 228 - 00 - BLU </td><td>側面に開けた穴</td></tr><tr><td>垂直ピペットプラー</td><td>デビッドKopfは楽器</td><td>モデル720 </td><td>セッティング：16.3（ヒーター）および2.3（ソレノイド） </td></tr><tr><td>ナトリウムホウケイ酸ガラス毛細管</td><td>サターのインストゥルメント</td><td> BF100 - 58から10 </td><td> （OD、1.0ミリメートル、ID 0.58ミリメートル、10 cmの長さ） </td></tr><tr><td> 1Xダルベッコ改変イーグル培地（DMEM） </td><td>インビトロジェン</td><td> 11995073 </td><td></td></tr><tr><td>ダルベッコ</td><td>インビトロジェン</td><td> 14190250 </td><td> pH7.4の</td></tr><tr><td>リン酸緩衝食塩水（D - PBS）（1X）、液体</td><td></td><td></td><td></td></tr><tr><td> EDTA 4NA、液体とトリプシン、0.05％（1X） </td><td>インビトロジェン</td><td> 25300054 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>インビトロジェン</td><td> 12483-020 </td><td>熱は、不活性化する</td></tr><tr><td>血球計</td><td> Hausserサイエンティフィック、VWR </td><td> 15170-090 </td><td></td></tr><tr><td>遠心</td><td>エッペンドルフモデル​​5810R </td><td> 5811 000.010 </td><td></td></tr><tr><td>細かい点鉗子</td><td> VWR </td><td> 25607-856 </td><td></td></tr><tr><td>タイゴンR - 3603チューブ</td><td> VWR </td><td> 63009-983 </td><td> 50フィート（1/32-inch内径、3/32-inch外径、1/32-inch壁の厚さ</td></tr><tr><td>注射18ゲージの針のための注射針</td><td> BD </td><td> 305195 </td><td> 100のボックス</td></tr><tr><td>注射の場合は1 mLシリンジ</td><td> BD </td><td> 309602 </td><td> 100のボックス</td></tr><tr><td>光ファイバ顕微鏡の照明</td><td> Amscope </td><td> HL250 - AY </td><td> 150W </td></tr><tr><td>キムワイプ</td><td> VWR </td><td> 10805-905 </td><td></td></tr><tr><td> V.有爪類の種子（カリフォルニア黒眼がない。5） </td><td>バーピー</td><td> 51771A </td><td></td></tr><tr><td>屋内成長が点灯</td><td>ホテルサンライト新宿、庭師の供給</td><td></td><td></td></tr><tr><td>メチル- PEO4 - NHSエステル</td><td>ピアース</td><td> PI22341 </td><td></td></tr><tr><td> MPEG - NHS、PEGスクシンイミジルエステル、MW 2000 </td><td> NANOCS </td><td> PEG1 - 0002 </td><td></td></tr><tr><td>のAlexa Fluor 647カルボン酸（スクシンイミジルエステル） </td><td>インビトロジェン</td><td> A20006 </td><td></td></tr><tr><td>オレゴングリーン488スクシンイミジルエステル* 6 -異性体* </td><td>インビトロジェン</td><td> O - 6149 </td><td></td></tr><tr><td>ジメチルスルホキシド（DMSO） </td><td>シグマ</td><td> D8418 </td><td></td></tr><tr><td>二塩基性リン酸一水素</td><td>シグマ</td><td> 379980 </td><td> K2HPO4（phopshateバッファ用） </td></tr><tr><td>一塩基性リン酸二水素</td><td></td><td> P5655 </td><td> KH2PO4（phopshateバッファ用） </td></tr><tr><td>スーパーローズ6サイズ排除カラム</td><td> GEヘルスケアバイオサイエンス</td><td> 17-0673-01 </td><td></td></tr><tr><td> AKTAエクスプローラ100クロマトグラフ</td><td> GEアマシャムファルマシア</td><td> WS - AKTA100 </td><td></td></tr><tr><td> AKTAハイフローキット</td><td> GEヘルスケアバイオサイエンス</td><td> 18-1154-85 </td><td></td></tr><tr><td>スクロース</td><td>シグマ</td><td> S0389 </td><td></td></tr><tr><td>超遠心分離機</tD&gt; <td>ベックマン</td><td></td><td></td></tr><tr><td> SW 28 Tiローター</td><td>ベックマン</td><td> 342204 </td><td>スイングバケット</td></tr><tr><td> 50.2 Tiローター</td><td>ベックマン</td><td> 337901 </td><td>固定角度</td></tr><tr><td>アミコンウルトラ- 15遠心フィルターユニット</td><td>ミリポア</td><td> UFC910008 </td><td> 100kDaのカットオフ</td></tr><tr><td>勾配前者</td><td>バイオラッド</td><td></td><td></td></tr><tr><td>デキストラン、フルオレセイン、70,000 MW、陰イオン性</td><td>インビトロジェン</td><td> D1823 </td><td></td></tr><tr><td>ディスク共焦点蛍光顕微鏡をスピニング</td><td>クォーラム、横河電機 CSU 10、横河</td><td> N / </td><td></td></tr><tr><td>落射蛍光広視野顕微鏡</td><td>クォーラム;ツァイスアクシオ審査、ツァイス</td><td> N / </td><td></td></tr><tr><td>浜松ImagEM 9100〜12 EM - CCDカメラ</td><td>クォーラム;浜松</td><td> N / </td><td></td></tr><tr><td>顕微鏡用温度のエンクロージャのユニット</td><td>精密プラスチック</td><td> N / </td><td></td></tr><tr><td>真空グリース</td><td> VWR </td><td> 59344-055 </td><td></td></tr><tr><td>円形のガラスはカバースリップしません。 1（18 mm）の</td><td> VWR </td><td> 16004-300 </td><td></td></tr><tr><td> Volocityソフトウェア</td><td>パーキンエルマー</td><td></td><td></td></tr><tr><td>ニワトリ胚のエンクロージャ</td><td></td><td>カスタムの作製</td><td></td></tr><tr><td>繊細なはさみ</td><td> VWR </td><td> 25608-203 </td><td></td></tr><tr><td>ホルマリン</td><td> Bioshop </td><td> FOR201.500 </td><td> fumehoodで使用</td></tr><tr><td>最適なカッティング</td><td>フィッシャー、ティッシュテック</td><td> 1437365 </td><td></td></tr><tr><td>温度（OCT） </td><td></td><td></td><td></td></tr><tr><td>プラスチック金型</td><td>フィッシャー</td><td> 22-038217 </td><td></td></tr><tr><td> VWR VistaVision HistoBond粘着スライド</td><td> VWR </td><td> 16004-406 </td><td></td></tr><tr><td> DAPIで金を延ばす</td><td>インビトロジェン</td><td> P36931 </td><td></td></tr><tr><td>クライオスタット用替刃</td><td>フィッシャー</td><td> 12-634-2 </td><td></td></tr><tr><td>クライオスタット</td><td>ライカCM 3050 S </td><td> 14047033518 </td><td></td></tr></table>

evaluation of nanoparticle uptake in tumors in real time using intravital imaging

このような超音波、MRI、PETとCTのような腫瘍イメージング、、のための現在の技術は、適切な異種移植モデルの有用性を強調し、微視的なレベル1,2,3での腫瘍のナノ粒子の取り込みの評価のために高解像度の画像を得ることができない詳細な取り込みの分析を実行するインチここで、我々は、修正された、シェルのないニワトリ胚モデルにおけるヒト腫瘍異種移植片でのナノ粒子の取り込みを評価するために高解像度の生体内イメージングを使用してください。それはヒトの腫瘍の成長をサポートしているため、ニワトリ胚のモデルは、特にin vivoでの解析でこれらのために適している、比較的安価であり、麻酔や手術は4,5は必要ありません。漿尿膜（CAM）6に移植するときに腫瘍細胞は7日以内に完全に血管移植片を形成する。結果として得られる腫瘍は、ホストまたは腫瘍どちらのシステムにおいてもほとんど影響を与えずに最大72時間まで維持することができる非侵襲的リアルタイム、高分解能のイメージングにより可視化。腫瘍への遠位投与サイズと処方の広い範囲でのナノ粒子は可視化し、血流を介してそれらの流れのように定量化することができる、漏れやすい腫瘍血管から浸出し、腫瘍部位に蓄積する。ここでは、近赤外蛍光色素および/ ​​またはポリエチレングリコールポリマー（PEG）7、8、9,10,11で飾らササゲモザイクウイルス由来のナノ粒子（CPMV）の分析について説明します。静脈内投与時には、これらのウイルス粒子が急速に外部の両方の血管系のグローバルなラベルで、腫瘍7,12内で、その結果、内皮細胞によって内在化されています。ウイルス粒子のPEG化は、それらの血漿中半減期を増加させ、循環で自分の時間を拡張し、最終的に強化された透過性と保持（EPR）効果7、10,11を介して腫瘍で、その蓄積を高める。腫瘍中のナノ粒子の蓄積の速度および程度は画像解析ソフトを用いて経時的に測定される。この手法は、ヒトの腫瘍にナノ粒子のダイナミクスを可視化し定量化するために、両方のメソッドを提供します。

Watch this Scientific Journal Video about Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging at JoVE.com

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

我々は、鳥類の胚のモデルでは動的な、リアルタイム生体イメージングを用いたヒト異種移植腫瘍の血管系でのナノ粒子の局在を定量化する新しいアプローチを提示する。

生体内イメージングを使ってリアルタイムで腫瘍におけるナノ粒子の取り込みの評価

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

evaluation-nanoparticle-uptake-tumors-real-time-using-intravital

Research

JoVE Journal

Medicine

18.2K ビュー.  University of Western Ontario. 我々は、鳥類の胚のモデルでは動的な、リアルタイム生体イメージングを用いたヒト異種移植腫瘍の血管系でのナノ粒子の局在を定量化する新しいアプローチを提示する。

ビデオ: Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

婦人科腫瘍におけるセンチネルリンパ節の術中検出のためのマルチスペクトルのリアルタイム蛍光イメージング

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

医学

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

定量的リアルタイムPCR（定量PCR）による前立腺腫瘍におけるマイクロRNAの検出

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

ラットの精巣挙筋の虚血再灌流障害における白血球 - 内皮相互作用のリアルタイムデジタルイメージング（IRI）

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

パフォーマンスのリアルタイムのフィードバックを使用して再教育運動

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

蛍光SAPC-DOPSナノベシクルを用いて脳腫瘍、関節炎のin vivo光イメージング中

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化学療法誘発性血管毒性 - リアルタイム<em&gt;インビボ</em船舶減損の&gt;イメージング

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

ヒト全血でのリアルタイム細胞毒性アッセイ

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

新生児マウスの肺における肺流体体積のリアルタイムX線イメージング

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

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

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

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

定量的リアルタイムPCRによるマウス乳腺腫瘍モデルにおける肺転移の評価

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

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...