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1。プロトコル<ol><li> 0.1 mgのケタミンと体重1グラム当たりキシラジンの0.01 mgの用量でケタミンおよびキシラジンでマウスを麻酔。カルプロフェン（体重1グラム当たり0.005 mg）を、鎮痛のために使用され、術前に投与される。 </li><li>歯科用ドリルビットを含む外科的なツールのすべては、オートクレーブで蒸気滅菌されています。バッチ手術が実行される場合には、手術器具の先端が、後続の各手術前にガラスビーズ滅菌器（ファイン科学ツールFST 250）で再滅菌しなければならない。 </li><li>マウスはつま先のピンチへの応答の有無によって評価麻酔の外科平面に達していると、それは、その後〜37℃の体温を維持するために加熱されたパッドの上に置かれる綿のパッド上に配置され麻酔の深さは、手術中に頻繁に監視されています。 </li><li>毛皮はほぼ三角形の面積の背側頭部〜鼻孔に尾3ミリメートルから削除され、CEに尾側に延びているrvical椎骨。眼軟膏は、脱水や刺激から守るために目に適用されます。 </li><li>マウスは腹recumbancyに配置され、皮膚を外科ヨウ素、70％エタノール、滅菌綿棒で消毒されています。頭蓋骨の前頭葉と頭頂骨の背側面を覆う皮膚の0.8×1.0 cm商品フラップは細かいハサミを用いて除去する。 </li><li>外用鎮痛、0.5％リドカイン、​​傷の周囲の骨膜と露出組織に局所的に適用されます。骨膜を穏やかにメスの刃で頭蓋骨を削って除去され、これが骨に付着するシアノアクリレート系接着剤を助けるでしょう。 </li><li>興味のある皮質領域はマーカーで頭蓋骨に概説されています。関心領域は、基礎となる大型船への損傷を避けるために、頭蓋骨の縫合線の上に位置するべきではありません。 </li><li>シアノアクリレート系接着剤の薄層を除いてserosanguinous流体の漏出を防止するために、創傷の縁に適用され、頭蓋骨のことです関心のある領域。 </li><li>光によって活性化された歯科用セメントは、頭蓋骨を封止するための我々のプロトコルで使用されています。歯科用セメントを塗布する前に、間伐のために意図された領域以外のすべての露出頭蓋骨をプライマーとし、続いて光活性歯科用セメントキットから接着剤で処理されます。接着剤が乾燥したら、光によって活性化された歯科用セメントは、頭蓋骨をカバーするために適用されます。その後の手術とイメージングのためのマウスの頭部を安定させるために、滅菌した＃00から90六角ナットを薄くする領域からの距離に歯科用セメントに埋め込まれています。光によって活性化された歯科用セメントの使用は、私たちは全体の手順を完了するために急いでしなくても、頭蓋骨を準備することができます。頭蓋骨に歯科用セメントの準備が完了すると、歯科用セメントは、可視光で硬化させる。 </li><li>歯科用セメントを硬化させた後、定位固定装置からヘッドピースホルダーは、＃00から90ネジを使用して、マウスの頭の上に六角ナットに取り付けられている。 </li><li> DR室温のop、0.9％滅菌生理食塩水を薄くするために頭蓋骨の領域に適用されます。実体顕微鏡の助けを借りて、高速マイクロドリルは、関心のある領域にわたって頭蓋骨の円形領域（通常は直径〜4,5 mm）を薄くするために使用されます。掘削と掘削面積に生理食塩水のアプリケーションは、基礎となる脳組織への熱損傷を避けるために、間伐手順の間に断続的に行われている。生理食塩水は、熱を吸収し、また、骨を柔らかくするのに役立ちます。 </li><li>マウスの頭蓋骨は海綿骨の厚い層を挟んで、緻密骨の2つの薄層を含む。緻密骨と海綿骨の大部分の外部層は、ドリルを使用して除去される。 </li><li>海綿骨の大部分が除去された後、海綿骨内の残りの空洞が掘削は内部コンパクト骨層に近づいていることを示し、解剖顕微鏡下で見ることができます。この段階では、頭蓋骨の厚さは依然として以上50μm以下である必要があります。間伐頭蓋骨は次のようになります非常に薄い（〜20μm）と滑らかな製剤を得るために慎重に続けた。 </li><li>頭蓋骨の所望の厚さ（〜20μm）を達成されたら、頭蓋骨が最初の約10分間、ダイヤモンドペーストを使用したカスタムメイドのシリコーンホイップ（6から15μm）を用いて研磨されています。ダイヤモンドペースト研磨は2つの目的があります。まず最初に、それはさらにそれによって、基礎となる脳組織への偶発的な損傷を回避すること、間伐頭蓋骨に圧力をかけることなく頭蓋骨を薄く。ファイングレイン·スズ酸化物は、さらに頭蓋骨の研磨に使用される前に、第二に、それは薄くなった頭蓋骨の初期研磨工程として機能します。 </li><li>ダイヤモンドペースト研磨後、薄くなった頭蓋骨は、約10分間酸化スズで研磨されています。頭蓋骨を研磨したら薄くなった頭蓋骨がきれいに表示されるまで、酸化スズは、生理食塩水で流されています。 </li><li>頭蓋窓が in vivoイメージングに縦に使用する場合は、このステップで、洗浄薄い頭蓋骨領域を乾燥させる。クリアの小滴シアノアクリレート系接着剤は頭蓋窓を密閉するために適用されます。間伐頭蓋骨とカバーガラスの間にシアノアクリレート系接着剤の層はできるだけ薄くする必要があります。 </li><li>注射はポート頭蓋ウィンドウの作成後に実行する必要がある場合は、この段階では、26ゲージの注射針は、洗練された薄頭蓋骨ウィンドウの片側に小さな開口部を作成するために使用されます。この開口はGBM細胞注入のために使用されます。 </li><li>直径約50μmを開く先端で滅菌ガラスピペット、ピペットプラーを使用してプルアップされています。このピペットはGBM細胞注入のために使用されるでしょう、そして、マイクロマニピュレータ上にロードされます。ピペットはGBM細胞注射部位は、最終的な撮像部位から離れるとなるように、30度の角度でロードされます。ピペットの先端は、解剖顕微鏡下で除去する。 GBMの細胞懸濁液をシリンジポンプを用いて吸引することによりピペットにフロントロードされます。 </li><li>注入用ピペットはGBM細胞がロードされたら、マウスが目の下で後方に移動さ電子顕微鏡。注入用ピペットは頭蓋窓にある小さい開口部に向かって下降し、最終的にマイクロマニピュレーターを用いて開口部を通って脳に挿入されます。 GBMの細胞懸濁液1μlを脳脳表面100から200ミクロン（：0.1μl/分、持続時間：10分回転数）に注入される。 </li><li>頭蓋骨を乾燥させ、明確なシアノアクリレート系接着剤の小滴は、乾燥頭蓋骨に適用され、3ミリサイズ＃1のカバーガラス片が薄く、洗練された頭蓋骨領域に接着されている。カバースリップと隣接する歯科用セメントの間の空間は、シアノアクリレート接着剤で封止されている。 </li><li>手術後、マウスは1ミリリットル暖かい滅菌生理食塩水を皮下注射していると完全に麻酔から回復するまで体温を維持するために補足的な暑さを備えている。 </li><li>イメージングのために、マウスは0.1 mgのケタミンと体重1グラム当たりキシラジンの0.01 mgの用量でケタミンおよびキシラジンで麻酔する。マウスはカスタム定位フレームを使用して固定化されている顕微鏡下で。マウスはケタミンの反復使用と依存の兆候を示すようにin vivoイメージングの繰り返しが日常的に行われている場合、イソフルランは、より良いオプションになります。 </li></ol> 2。代表的な結果成功したポート頭蓋窓手術は頭蓋窓が数週間から数ヶ月のための明確なままにすることができます。後方散乱光を用いて可視化として、 図1はポート頭蓋窓の下に血管系を示しています。これらの血管系の画像は、繰り返しイメージングのための同じ脳領域を特定するための目印として用いた。血管系の内皮細胞およびGBM細胞の両方を可視化するために、我々はB6.Cg-GT（ROSA）26Sor TM9（CAG-tdTomatoと血管内皮細胞をラベルするB6.Cg-TG（テック-CRE）1Ywa / Jマウスを、交差させる）Hze / Jマウス、および赤色蛍光タンパク質tdTomatoで標識された血管内皮細胞と生成されたマウス。そして、私たちは、これらマイル上のポートの開頭手術を行ったCE、および頭蓋窓の側の小さな開口部を通って、自分の脳にGFP標識GBM細胞を注入した。次に、注射したマウスで縦方向に二光子イメージング生体内で行った。レーザー励起波長は、GFPとtdTomato両方の励起につながっ910 nmで設定されていました。二光子イメージングは同時赤色/緑色蛍光検出能力のための2つの検出器で特注の二光子レーザー走査顕微鏡で行った。 図2は、血管系の近くに注入されたGBM細胞から神経膠腫開始の in vivoでの例に示します。ポート頭蓋窓もin vivoイメージングタイムラプス慢性ための優れた選択肢です。 図3に示すポートの手術をせずにマウスの皮質切片のGFAP染色、ポート手術後のマウスの7日間、マウス7日、次のポートの手術と生理食塩水を注入。それは手術をせずにマウスとPOR付マウスその画像からも明らかであるTSの手術は脳内浸透と生理食塩水の注射をピペットに起因し、生理食塩水注射後のに対し、グリオーシスは、マウスの脳組織で観察される、グリオーシスを持っていない。この結果は、神経膠症は頭蓋窓は、ここで説明ポートが一般的に&quot;開かれた頭蓋骨&quot;慢性頭蓋ウィンドウに関連付けられている神経膠症につながらないことを独立した検証を提供して、ポート頭蓋窓の下では発生しませんを示しています。 図4は高解像度を示していますPortsウィンドウもin vivoイメージングの高分解能のための優れた選択肢であることを実証して手術の日に同じマウス（0日目）からの樹状突起スパインのイメージングと32日Portsウィンドウを作成した後（32日目）。 <img alt="図1" src="/files/ftp_upload/4201/4201fig1.jpg" /> 図1：後方散乱光を用いて研磨し、補強された間伐頭蓋骨頭蓋窓下の血管系の可視化。日tを表す<html>手術の数日後、彼は数、画像はすぐにGBM細胞注入が完了した後に手術の日に始まる撮影された。スケールバー：0.5mmの<a href="http://www.jove.com/files/ftp_upload/4201/4201fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img alt="図2" src="/files/ftp_upload/4201/4201fig2.jpg" /> 図2：in vivoで注入されたGBM細胞と血管系の可視化。 TEK-CREおよびROSA26 CAG-tdTomato両方を持つマウスは、GBM細胞注入のために使用された。血管内皮細胞は、tdTomato（赤）で標識した、とGBM細胞は、GFP（緑色）で標識した。画像はGBM細胞注射後24時間を開始を取得した。ビューの10-12隣接するフィールドの合計を採取し、最終的な画像を生成するために、合計フィールドの中央に注入された識別されたGBM細胞と、互いに隣接して組み立てた。日は、手術後の日数を表す。スケールバー：100μmである。href =の&quot;http://www.jove.com/files/ftp_upload/4201/4201fig2large.jpg&quot;ターゲット= &quot;_blank&quot;&gt;拡大図を表示するには、ここをクリックしてください。 <img alt="図3" src="/files/ftp_upload/4201/4201fig3.jpg" /> 図3：グリオーシスは、ポート頭蓋窓手術後は発生しません。アッパーパネル：低倍率下でのマウス脳冠状断面。左のパネル：任意の頭蓋窓の手術をせずにマウス;なしGFAPの活性化は、脳組織で観察される。中央のパネル：脳の右側で生成されたポート頭蓋窓が付いているマウス、ノーGFAPの活性化は、脳組織で観察される。右のパネル：Portsウィンドウの側からポート頭蓋窓と生理食塩水の注射でマウス。 GFAPの活性化は、Portsウィンドウの側ではなく、無傷の脳組織の側に観察される。スケールバー：1ミリメートル。下部パネル：上部パネル画像のホワイトボックスに示されているように地域での脳組織の高出力画像。スケールバー：100μmである。 <ahref =の&quot;http://www.jove.com/files/ftp_upload/4201/4201fig3large.jpg&quot;ターゲット= &quot;_blank&quot;&gt;拡大図を表示するには、ここをクリックしてください。 <img alt="図4" src="/files/ftp_upload/4201/4201fig4.jpg" /> 図4高解像度THY-1 YFPHマウス由来の樹状突起スパインのイメージング。画像は、ポートの手術の日に取得され、32日ポートの手術後に行った。それは、ほとんどの棘が一日に提示したイメージから、0ポート手術後のはっきり見える32日アールは明らかである。スケールバー：2μmである。

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成功したポート頭蓋窓の鍵は、間伐や研磨です。間伐初期は迅速に行うことができますが、注意が広い面積にわたって頭蓋骨の間伐均質性を確保するために取られるべきである。我々は一般的に生理食塩水がマイクロドリルは一度頭蓋骨を通過した直後に蒸発するような、頭蓋骨に生理食塩水の薄い層を適用し、薄い頭蓋骨ワンパス時。これはゆっく​​りと、しかし均一さらに薄い頭蓋骨?...

<table border="1"><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>ピルビン酸ナトリウムDMEM/F12（1:1） </td><td>サーモサイHyclone社</td><td> SH3026101 </td><td></td></tr><tr><td> B-27に、無血清サプリメント（50×） </td><td>インビトロジェン</td><td> 17504-044 </td><td></td></tr><tr><td>グルタ1補助</td><td>インビトロジェン</td><td> 35050061 </td><td></td></tr><tr><td>ペニシリン - ストレプトマイシン溶液</td><td>サーモサイHyclone社</td><td> SV30010 </td><td></td></tr><tr><td> Accutase酵素細胞分離培地</td><td> eBiosciences </td><td> 00-4555-56 </td><td></td></tr><tr><td> T25フラスコ - ベントキャップグリーン</td><td>ザルスタット</td><td> 83.1810.502 </td><td></td></tr><tr><td> 70％アルコール</td><td> JAX LAHS </td><td></td><td></td></tr><tr><td> 10パーセントポビドンヨード局所用溶液</td><td> JAX LAHS </td><td></td><td></td></tr><tr><td>ケタミン塩酸</td><td>バトラーアニマルヘルスサプライ</td><td> NDC＃11695-0550-1 </td><td></td></tr><tr><td>キシラジン</td><td> Akorn株式会社</td><td> NADA＃139から236 </td><td></td></tr><tr><td>カルプロフェン</td><td> JAX LAHS </td><td></td><td></td></tr><tr><td>眼軟膏</td><td> Dechra獣医製品</td><td> 17033-211-38 </td><td></td></tr><tr><td> 0.5パーセントリドカイン塩酸</td><td> REGENT株式会社</td><td> NDC 0517-0625-25 </td><td></td></tr><tr><td>シアノアクリレート接着剤</td><td>ヘンケル社</td><td> 46551 </td><td></td></tr><tr><td>滅菌綿棒</td><td>フィッシャー·サイエンティフィック</td><td> 23から400から114 </td><td></td></tr><tr><td>ダイヤモンドペースト</td><td>ウィジェットサプライ</td><td> BBE60 </td><td></td></tr><tr><td>酸化スズ</td><td> LORTONE株式会社</td><td> 591から038 </td><td></td></tr><tr><td>ライナーボンド2V </td><td>クラレメディカル株式会社</td><td> 1921-KA </td><td></td></tr><tr><td>クリアフィルAP-X </td><td>クラレメディカル株式会社</td><td> 1721-KA </td><td></td></tr><tr><td>生理食塩水</td><td> JAX LAHS </td><td></td><td></td></tr><tr><td>カバーガラス</td><td>ワーナー·インスツルメンツ</td><td> 64から0720 </td><td></td></tr><tr><td>六角ナット</td><td>小さなパーツ株式会社</td><td> HNX-0090-C </td><td></td></tr><tr><td>注射器ポンプ</td><td> Syringepump.com </td><td> NE-1000 </td><td></td></tr><tr><td>二光子イメージングシステム</td><td>カスタム構築された（任意の商用システムがそう仕事） </td><td></td><td></td></tr></table>

imaging glioma initiation in vivo through a polished and reinforced thin-skull cranial window

神経膠腫は、ヒト癌の最も致命的な形態の一つです。日付手術に最も効果的な神経膠腫の治療は、放射線に続くほとんどの患者は神経膠腫の再発1,2に起因診断後五年以上生きていけないように、患者にわずかな利益を治療しています。ヒト脳腫瘍における癌幹細胞の発見は、神経膠腫の3のための新規治療戦略の開発に多大な影響を持っていることのための可能性を秘めています。がん幹細胞は、その能力、自己再生すると区別するために、新たな腫瘍4を開始する能力を持っている腫瘍で唯一の細胞であると考えられているの両方で定義されています。神経膠腫再発以下の放射線療法は、治療へのグリオーマ幹細胞の抵抗（GSCs）から発生すると考えられている5-10。 生体内では 、GSCsは、その幹細胞様の特性11-14を維持するために重要である血管周囲のニッチに存在することが示されている。 organiに中央GSCニッチのzationは12血管内皮細胞である。既存のエビデンスは、血管内皮細胞とGSCsとそれらの相互作用が腫瘍の開発のために重要であり、神経膠腫のための重要な治療標的としての内皮細胞とGSCsとそれらの相互作用を識別することを示唆している。 GSCsの存在は、同所移植15時に新しい腫瘍を開始するために彼らの能力によって実験的に決定される。これは典型的には深刻な免疫不全マウスの、またはホストコンジェニックマウスの脳にマウスGBM細胞の脳にヒト腫瘍から単離されたGBM細胞の特定の数を注入することによって達成される。腫瘍の成長のためのアッセイはその後注入GBM細胞間GSCsが新しい腫瘍 - 典型的には数週間あるいは数ヶ月を生じさせることができるのに十分な時間後に実行されます。したがって、既存のアッセイは、in vivoでのシングルGSCsから腫瘍の開始の重要な病理学的過程の検査を許可していません。したがって、本質的なIGSCsと腫瘍の開始の初期段階では血管内皮細胞との相互作用の具体的な役割にnsightsが不足している。このような洞察は、神経膠腫の新しい治療戦略を開発するために重要であり、患者では神経膠腫の再発を防止するための大きな意味を持つでしょう。ここでは、ポートの頭蓋のウィンドウプロシージャは16を適応していると、生体内で二光子顕微鏡は、生きたマウスの脳内に注入されたGBM細胞から腫瘍の開始の可視化を可能に。我々の技術は、神経膠腫の開始時GSCsと血管内皮細胞との間の重要シグナル伝達機構を解明するための今後の取り組みへの道を開くでしょう。

Watch this Scientific Journal Video about Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window at JoVE.com

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

ポリッシュと補強薄頭蓋骨（ポート）頭蓋窓と神経膠芽腫（GBM）の細胞注入を組み合わせることによって、我々は、神経膠腫の開始と縦ライブマウスの脳内に注入GBM細胞からの成長を観察することができます。

イメージンググリオーマ開始<emインビボで&gt;</em&gt;ポリッシュと補強シン頭蓋骨頭蓋窓スルー

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

imaging-glioma-initiation-vivo-through-polished-reinforced-thin-skull

Research

JoVE Journal

Medicine

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

13.0K ビュー.  The Jackson Laboratory. ポリッシュと補強薄頭蓋骨（ポート）頭蓋窓と神経膠芽腫（GBM）の細胞注入を組み合わせることによって、我々は、神経膠腫の開始と縦ライブマウスの脳内に注入GBM細胞からの成長を観察することができます。

ビデオ: Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

炎症性プラークの赤外蛍光（NIRF）血管内分子イメージング、マルチモーダルアプローチの近くin vivoでの動脈硬化のイメージングに

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

医学

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

生体デュアル基質生物発光イメージングにおける

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

エンドセリン-1誘発性中大脳動脈閉塞中大脳動脈を可視化するために頭蓋窓を利用

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration of specific neural processes in the central nervous system. However, information is lacking on the molecular impact of these changes, especially on the endogenous opioid system during migraine headaches, and neuronavigation through these changes has never been done. This study aimed to investigate, using a novel 3D immersive and interactive neuronavigation (3D-IIN) approach, the endogenous &#181;-opioid transmission in the brain during a migraine headache attack in vivo. This is arguably one of the most central neuromechanisms associated with pain regulation, affecting multiple elements of the pain experience and analgesia. A 36 year-old female, who has been suffering with migraine for 10 years, was scanned in the typical headache (ictal) and nonheadache (interictal) migraine phases using Positron Emission Tomography (PET) with the selective radiotracer [11C]carfentanil, which allowed us to measure &#181;-opioid receptor availability in the brain (non-displaceable binding potential - &#181;OR BPND). The short-life radiotracer was produced by a cyclotron and chemical synthesis apparatus on campus located in close proximity to the imaging facility. Both PET scans, interictal and ictal, were scheduled during separate mid-late follicular phases of the patient's menstrual cycle. During the ictal PET session her spontaneous headache attack reached severe intensity levels; progressing to nausea and vomiting at the end of the scan session. There were reductions in &#181;OR BPND in the pain-modulatory regions of the endogenous &#181;-opioid system during the ictal phase, including the cingulate cortex, nucleus accumbens (NAcc), thalamus (Thal), and periaqueductal gray matter (PAG); indicating that &#181;ORs were already occupied by endogenous opioids released in response to the ongoing pain. To our knowledge, this is the first time that changes in &#181;OR BPND during a migraine headache attack have been neuronavigated using a novel 3D approach. This method allows for interactive research and educational exploration of a migraine attack in an actual patient's neuroimaging dataset.

3D-Neuronavigation In Vivo Through a Patient&#039;s Brain During a Spontaneous Migraine Headache

In this study, the authors report for the first time a novel 3D-Immersive &#38; Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the &#956;-opioid system of a patient's brain in vivo.

3D-ニューロ<em&gt;インビボ</em&gt;自発片頭痛時には、患者の脳を通じ

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

シーケンシャル<em&gt;インビボ</em&gt;骨折修復中に、骨形成幹細胞/前駆細胞のイメージング

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

合わせた<em&gt;インビボ</em&gt;光とμCTイメージングマウスにおける整形外科インプラント感染で感染症、炎症、および骨解剖学を監視する

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

State of the Art Cranial Ultrasound Imaging in Neonates

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a patient is unstable. In addition it is radiation-free and allows serial imaging. CUS possibilities have steadily expanded. However, in many neonatal intensive care units, these possibilities are not optimally used. We present a comprehensive approach for neonatal CUS, focusing on optimal settings, different probes, multiple acoustic windows and Doppler techniques. This approach is suited for both routine clinical practice and research purposes. In a live demonstration, we show how this technique is performed in the neonatal intensive care unit. Using optimal settings and probes allows for better imaging quality and improves the diagnostic value of CUS in experienced hands. Traditionally, images are obtained through the anterior fontanel. Use of supplemental acoustic windows (lambdoid, mastoid, and lateral fontanels) improves detection of brain injury. Adding Doppler studies allows screening of patency of large intracranial arteries and veins. Flow velocities and indices can be obtained. Doppler CUS offers the possibility of detecting cerebral sinovenous thrombosis at an early stage, creating a window for therapeutic intervention prior to thrombosis-induced tissue damage. Equipment, data storage and safety aspects are also addressed.

Cranial ultrasound (CUS) is a valuable tool for brain imaging in critically ill neonates. This video shows a comprehensive approach for neonatal (Doppler) CUS for both clinical and research purposes, including a bedside demonstration of the technique.

新生児におけるアート頭蓋超音波画像の状態

Cranial ultrasound (CUS) is a reputable tool for brain imaging in critically ill neonates. It is safe, relatively cheap and easy to use, even when a ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

マウス収縮圧力を測定するために子宮内テレメトリ<em&gt;インビボ</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

馬腱でテクネチウム-99m標識骨髄間葉系幹細胞のin vivoイメージングと追跡中

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...