私たちは、博士を認める。カミッロリコルディ、アントネッロピレッジ、R.ダマリスMolano、ステファンSpeierと実りある議論のためのダニエルNyqvist。我々はまた、ビデオ録画のヘルプはEleutヘルナンデスとディエゴ·エスピノーサ-Heidmann技術支援のために、マイク·バルデスとマーガレットFormosoに感謝します。バイロンマルドナドは、編集された記録され、最終的な映像を作り出した。研究支援は、糖尿病研究所財団（提供された<a href="http://www.DiabetesResearch.org" target="_blank">www.DiabetesResearch.org</a> ）、NIH / NIDDK / NIAID（MHAにF32DK083226、ACへのNIH RO3DK075487; PO.B.へU01DK089538）。 PO.Bに追加の研究支援はカロリンスカ研究所、スウェーデン研究評議会、スウェーデン糖尿病財団、ファミリーErling Perssonさん、財団、ファミリークヌートとアリスウォレンバーグ財団、スカンディア·インシュアランス·カンパニー·リミテッド、活気のある（から資金を介して提供されましたFP7-228933から2）、カロリンスカ研における糖尿病における研究戦略推進プログラムitutet、ノボノルディスク財団、バースフォンKantzow財団。

次の手順は2ステップで実体鏡下で行われ、最初のステップは、カニューレ内に島をロードする必要が第二段階では、ACEへの実際の移植である。動物で実行されるすべての手続きは、マイアミ大学の制度的動物のケアと使用委員会（IACUC）によって承認された。 1。移植するためのカニューレでロードランゲルハンス島<ol><li>サークルを絞り込むのに皿を回転させて中央培養皿の中の小島。 </li><li> &quot;リザーバ&quot;からカニューレを外し、清潔な表面にカニューレと接続チューブを配置します。リザーバは、フィルタ（ 図1a）を使用せずに300μlの使い捨てプラスチックピペットチップで作ることができる。 </li><li>リザーバ内に吸引時の小島の連続的な流れを確保するために貯水池の気泡を洗い流す。貯水池のフラッシングがフットペダルを使用してハンズフリー電動注射器ドライバを前方に駆動することによって行われます（ 図1b、c）である。また、これは（例えば生理食塩水、PBSまたは培養培地などの滅菌液であらかじめロード）リザーバーに膵島の吸引を可能にするために、シリンジ内の空間を作ります。 </li><li>優しくリザーバーに膵島の所望量を吸引除去する。彼らはリザーバーを入力して、底部に向かって一緒に残るような島は渦になる傾向がある。吸引は、フットペダルを使って後方電動注射器ドライバを駆動することによって行われます。 </li><li>接続チューブを介してリザーバにカニューレを再接続します。 </li><li>培養皿に戻ってカニューレ先端を配置し、カニューレ内に次にチューブに貯水池の小島を洗い流す。あなたは優しく（タッピング）チューブ（ 図1d） &quot;フリック&quot;でチューブ/カニューレを埋戻しとして膵島一緒残ることを確認します。先に島の全ての気泡がカニューレをフラッシュされる前または後に停止します。よくわからない場合は、小島が残って空気としてカニューレの奥に入ると停止前方に小島の泡は、ACEの小島からの逆流（逆流）を防止することができます。一夜消費します。 </li><li>この段階では、ACE（次の手順を参照してください）​​に膵島を注入する準備が整いました。 </li></ol> 2。前眼房に膵島移植<ol><li>ステレオスコープの下に暖かいパッドの上に麻酔マウスを置きます。 </li><li>酸素/イソフルレン麻酔器に接続されている麻酔 &quot;マスク&quot;にマウスの鼻を置きます。マスクは1ミリリットル使い捨てプラスチックピペットチップ（フィルターなし）から作られ、狭い方の端（ 図2a、b）を介して麻酔のチューブに接続されています。 </li><li>そっと眼の蓋が良い露出と簡単にアクセスできます（ 図2c）に目を光らせて空いている方の手の人差し指と親指を使用していて、 &quot;ポップ&quot;移植する引っ込める。これは上の過度の圧力によって、マウスの呼吸を妨げることなく完璧にいくつかの練習が必要になります首や頭への血流を遮断する。 </li><li>使い捨てインスリン注射器（29 - 31G）を使用して、メスのように、慎重に角膜の先端のみを貫通し、単一横切開を行います。 ACE（ 図2d）のうち、注入中の膵島の逆流を最小限にするために、角膜と角膜輪部の頂点との中点で切り込みを入れる。 </li><li>注意深く切開を通してカニューレを（小島がプリロード）を挿入します。 </li><li>ゆっくりとカニューレの膵島を取り出して虹彩の上にそれらを預ける。 ACEの過度の圧力上昇に起因する膵島逆流を防ぐために、切開に象限の反対の中でできるだけ少ないボリューム（s）として、短時間の推力で膵島を取り出します。これは、さらにカニューレを（ステップ1.6を​​参照してください）​​ロード中にチューブに膵島を圧縮することにより確保することができる。 </li><li>ゆっくりACEのカニューレを引っ込める。島の大ボリュームは圧力に起因する膵島還流として注入した場合、これは特に、重要なステップです必ずACEの内部に蓄積避けられないかもしれない。カニューレの周りに切開して過剰圧力を解放するために、ACEの中にある状態で膵島逆流を最小限に抑える/排除するために、穏やかにカニューレを回転させます。あなたはカニューレを撤回し、必要に応じて、ACEから完全に後退する前に圧力がおさまるまでカニューレを待つように試みると同時に逆流の兆候がないか確認してください。 </li><li>滅菌PBSまたは生理食塩水を移植された眼をすすぐ。 </li><li>最初の48時間、術後鎮痛（0.05 mg / kgの皮下）にブプレノルフィンを注入します。 </li><li>直ちに移植後の移植眼にエリスロマイシン点眼抗生物質軟膏を塗る。 </li><li>麻酔からの回復を可能にするために温めておいたケージに戻って動物を置きます。 </li></ol>

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PO.B.商用サービスのプラットフォームとして眼の前房を使おうとしているバイオテクノロジー会社Biocrine、の創設者の一人です。 ACはこの技術を保護する特許になります。

マウス膵島は、以前説明したように33、密度勾配上での精製に続くコラゲナーゼ消化を用いて単離した。孤立した膵島を移植する前に、一晩培養した。これは必要とされないかもしれないが、それは小島が単離手順から回復できるようにすることをお勧めします。これは、生き残った/堅牢な膵島の移植性を確保するように移植が糖尿病のレシピエントで実行される時に重要です。 ...

生体顕微鏡の進歩は、in vitro試験1で予測されていない生理現象を明らかにした。これは、生きている動物にインビトロ法によって得られた従来の知見を翻訳に挑戦を強調しています。過去10年間では、生きた動物の組織の可視化はかなりの撮像モダリティ2、3、4、5、6の技術の進歩によって改善された。これは、非侵襲的に標的組織の縦可視化を可能にするための実験動物モデルで実現可能なアプリケーションを用いた in vivoイメージングアプローチのための必要性に拍車をかけている。 このような磁気共鳴イメージングと陽電子放射断層撮影法、または生物発光イメージング技術としては、体7-8、9の奥深くの臓器/組織の非侵襲的イメージングを有効にしている。しかし、これらの技術は使用Oにもかかわらず、高いバックグラウンド信号と低空間分解能に起因する単一細胞の解像度を達成することはできませんF高コントラスト材料または組織特異的発光4。これは二光子蛍光共焦点顕微鏡10の出現で対処されました。二光子顕微鏡は、前例のない詳細11、12と携帯電話のイベントを可視化し、定量化するために生体内イメージングの研究を可能にしました。これは、健康と病気13、14、15、16の重要な生物学的プロセスの特性評価につながっている。先駆的な生体内イメージング研究は、主に切除された組織（ 例えば、リンパ節） を用いたin vivo条件で &quot;模倣&quot;しているが、他の研究では 、in situ 17、18、19、20、21 に画像さらさ標的組織への侵襲的なアプローチを使用しています。他の研究でも侵襲的なアプローチおよび in vivo 22、23、24、25 に制限された撮像解像度に関連付けられた制限を回避するには、 &quot;ウィンドウ室モデル&quot;を使用しています。ウィンドウ室モデルでは、透明な窓を有する室は、外科的にdiffeで皮膚に注入される動物の家賃の場所（背や耳、皮膚、乳房脂肪、肝臓など）（ 例えば 、マウス、ラット、ウサギ）。このアプローチは明らかにin vivoイメージングの高解像度を可能にしますが、それは、チャンバを移植する侵襲的手術を必要とし、数週間または数ヶ月22の上に縦イメージング研究を収容することができない場合があります。 それが最近になって前眼房（ACE）への移植、すなわち、低侵襲で高解像度の共焦点顕微鏡法を組み合わせることで、in vivoイメージングプラットフォーム26,27 における強力で汎用性の高いような&quot;自然な身体の窓&quot;を提供している。実証されたACEへの移植は組織28、29、30の様々な生物学的側面 ​​を研究するために、過去数十年で使用されてきた、そして高分解能イメージングとその最近の組み合わせにより、単一細胞の解像度で膵島の生理学を勉強して有効になって非侵襲的かつ長手方向に&lt;&gt; 26、27（商標）。このアプローチは、動物モデルにおける1型糖尿病（未発表データ）の開発の間に自己免疫応答を研究するために使用されました。また、それぞれのACE膵芽または個々の腎糸球体、（未発表データ）に移植することにより、腎機能の研究では、膵臓の発達を研究するだけでなく、ために使用された。このアプローチを使用した最近の報告書はさらに、膵島移植31日後の免疫応答を研究するための応用を実証しました。重要なのは、この研究では、眼の前房への移植は、実行する自然な身体のウィンドウを提供示した：in vivoで移植組織の（1）の長手方向の、非侵襲的イメージング、（2） 生体内でで 、細胞の表現型と生存率を評価するためにcytolabeling その場で 、標的組織で免疫細胞の浸潤（3）リアルタイム追跡、および局所適用または眼内注射によって（4）地元の介入。 ここでは、D膵島を用いて眼の前房に移植を行う方法emonstrate。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 説明/コメント </td></tr><tr><td> IsoTHESIA（イソフルラン） </td><td> Buttlerアニマルヘルスサプライ</td><td> 11695-6775-2 </td><td> 99.9パーセントイソフルラン/ mlの</td></tr><tr><td> Ketaset（ケタミンHCL） </td><td>フォートダッジ·アニマルヘルス</td><td> 0856-2013-01 </td><td>代替注射麻酔</td></tr><tr><td> Beprenex（ブプレノルフィン塩酸塩） </td><td>レキットベンキーザー·ヘルスケア（英国）株式会社</td><td> 12496-075-7-1 </td><td> 0.3 mg / mlの</td></tr><tr><td>エリスロマイシン眼軟膏、USP、0.5％ </td><td>アクロン</td><td> 17478-070-35 </td><td>移植された眼に予防的に適用された</td></tr><tr><td> 0.9％塩化ナトリウム（生理食塩水） </td><td>ホスピーラ社</td><td> 0409-7983-03 </td><td>静脈注射用。無菌の</td></tr><tr><td> PBS </td><td>ギブコ</td><td> 10010-023 </td><td> 1X。無菌の</td></tr><tr><td> CMRL培地1066 </td><td> Cellgro </td><td> 98から304-CV </td><td>補足し、CITの修正。小島のための好ましいメディア</td></tr></tbody></table>

transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time

生体内イメージングは​​、生物学の研究に不可欠なツールとして浮上している。その過程で、多くのイメージング技術は、非侵襲的に動物に異なる生物学的過程を研究するために開発されてきた。しかし、既存の生体内イメージングモダリティの主要な技術的な限界は、単一セルの解像能力を持つ非侵襲的、縦画像を組み合わせることができないことである。我々は前眼房への移植は、 生体内で細胞レベルの分解能で非侵襲的な、縦撮影が可能多目的実験プラットフォームを提供するなどの重要な制限を回避する方法をここに示す。我々は、マウスに移植手順を示し、すなわち、臨床的意義、膵島移植でモデルを使用して代表的な結果を提供します。前眼房に移植し、様々な組織で直接可視化を可能にするだけでは、このアプローチはがれするためのプラットフォームを提供長期的なフォローアップを実施し、標的組織で監視することにより、n個の薬。なぜなら目の給付だけでなく、移植治療の前房にその汎用性、組織/細胞移植のために、それは、このようなシグナル伝達とがんや自己免疫疾患の発症などの生理学的および病態生理学的プロセスを研究するために 、in vivoアプリケーションで他にも及ぶ。

&quot;良い​​&quot;移植を定義するいくつかのパラメータがあります。良い移植は出血せずに進み、映像に見られるように切開を行ったものです。出血は、ACE（ 図3a）にメス（針）の先端のみを貫通することによって防止/最小化されます。これはまた、虹彩の接触および穿刺を防ぐことができます。また、時間（ 図3c、d）の上に、角膜の濁りを発生させることなく、...

Watch this Scientific Journal Video about Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time at JoVE.com

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

眼内移植し、共焦点顕微鏡を組み合わせた新しいアプローチがグラフトされた組織内での単一セルの解像度で縦、非侵襲性のリアルタイムイメージングを可能にする<em生体内で&gt;</em&gt;。我々は、マウスの眼の前房に膵島を移植する方法を示します。

縦用の眼の前房に移植、非侵襲<emインビボ&gt;</emリアルタイムでのシングルセル分解能&gt;イメージング

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

transplantation-into-anterior-chamber-eye-for-longitudinal-non

Research

JoVE Journal

Medicine

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

13.3K ビュー.  University of Miami Miller School of Medicine. 眼内移植し、共焦点顕微鏡を組み合わせた新しいアプローチがグラフトされた組織内での単一セルの解像度で縦、非侵襲性のリアルタイムイメージングを可能にする。我々は、マウスの眼の前房に膵島を移植する方法を示します。

ビデオ: Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

高周波高分解能心エコー検査：虚血 - 再灌流マウス心臓における心筋ストレイン、収縮性、および僧帽弁閉鎖不全の非侵襲的な反復測定に関する最初の証拠

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

医学

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 ...

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

使用したラット腎移植後の急性拒絶反応の非侵襲的イメージング<sup&gt; 18</sup&gt; F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

ブラインドのナビゲーションスキルを支援するためにオーディオベースのバーチャル·ゲーム環境の開発

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

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 ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

生きたマウスのシリアル内視鏡イメージングを使用して、腸の異常のための新しい治療の有効性の非侵襲的評価

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

移植のためのカプセル化サーモ前脂肪細胞の脂肪組織デポへ

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

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 ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

縦<em&gt;インビボ</em&gt;脳血管系のイメージング：中枢神経系疾患との関連

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

再門脈部分肝移植のラット体内血管指向 70% 肝切除

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...