我々は、GL261 - lucの細胞の調製に関する有用な提案のためのレンチウイルスのシステムだけでなく、Mahilラオのためのプラスミドの寛大な贈り物のために博士ジョシュアB.ルービンに感謝します。 我々は、脳腫瘍研究（SSBTR）、バロー神経財団とその寛大な支援のためのウォレス財団を支援する学生に感謝する。 動物実験は、セントジョセフ病院と医療センターの動物実験使用の委員会が定めるガイドラインおよび規則に従って行った。

<ol>
	<li>Candolfi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathological+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.</a> J Neurooncol. 85, 133-148 (2007).</li><li>Stafford, P., Abdelwahab, M. G., do, K. i. m., Preul, Y., Rho, M. C., M, J., Scheck, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ketogenic+diet+reverses+gene+expression+patterns+and+redudes+reactive+oxygen+species+levels+when+used+as+an+adjuvant+therapy+for+glioma.">The ketogenic diet reverses gene expression patterns and redudes reactive oxygen species levels when used as an adjuvant therapy for glioma.</a> Nutr Metab. 7, (2010).</li><li>Caliper Life Sciences, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Living Image Software Version 4.0. VivoVision Systems. , (2010).</li><li>Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Jouanneau, E., Poujol, D., Gulia, S., Le, M. I., Blay, J. Y., Belin, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+are+essential+for+priming+but+inefficient+for+boosting+antitumour+immune+response+in+an+orthotopic+murine+glioma+model.">Dendritic cells are essential for priming but inefficient for boosting antitumour immune response in an orthotopic murine glioma model.</a> Cancer Immunol Immunother. 55, 254-267 (2006).</li></ol>

この記事へのフリーアクセスは、キャリパーライフサイエンスが主催している。

細胞の播種は、0.4mmのポケットを作成した後、脳の表面から2.6 mmの深さで注入される。針の適切な配置と深さを確保するためにX線は、C -アームまたは類似のX線画像の激化デバイスを使用して取得できますが、これはオプションです。動物が適切に鎮静されていない場合、手術の合併症は、動物の細胞の注入の間に移動できる点で、発生する場合があります。これは、針のトラックからの細胞の混合または出血の漏洩を引き起こす可能性があります。セルのリーク電流は、腫瘍細胞の異所性増殖を引き起こす。またburrholeはプロトコル4で説明されている2.3ミリメートルの内側行われた場合に行うことができます心室ではない穿刺することが重要です。針と細胞拡散の適切な配置は、2μlのメチレンブルー色素でマウスを注入し、注入された色素の場所を確認するために脳組織を解剖することによって試験した。 このプロトコルでは、in vivoイメージングシステムとt で IVISスペクトラムを使用している彼はこの楽器で使用するために設計されたイメージのソフトウェア（V 4.0）リビング。 （キャリパーライフサイエンス）。 in vivoイメージングシステムと画像解析ツールであらゆる匹敵するが、同様の結果を得るために使用することができます。これらのシステムは、実験的な頭蓋内腫瘍の成長をフォローするために従来の磁気共鳴画像（MRI）に比べていくつかの利点を提供します。最も明白な2楽器の相対的なコストである-動物MRI装置は、はるかに高価であり、一般的に熟練したMRI技術者のサービスを必要とする。ここに記載されたものとin vivoイメージングでは 、エンドユーザが行うことができます。 MRIデータの定量には時間がかかり、やや不正確である間の生物発光データは、定量的である。また、MRI画像は腫瘍細胞に加えて、浮腫や炎症を表示し、それが治療効果から腫瘍を分離することが困難な場合があります。これらの理由から成長する腫瘍の正確な体積測定値を得ることが困難な場合があります。生物発光は、ATPが必要ですしたがって、唯一の生きている腫瘍細胞は、腫瘍の大きさのデータに貢献する。マシンが使用可能であればそれにもかかわらず、MRIにはいくつかの利点があります。細胞は、MRIで可視化するために生物発光マーカーで標識する必要はありません。周囲の腫瘍浮腫を視覚化する能力は、いくつかの実験的なプロトコルのための利点があります。両方の技術の強さは、いずれかを使用すると、他の使用を排除しない、両方のテクノロジが利用可能になったとき、そのデータは腫瘍の成長だけでなく、同​​じ動物からペリ腫瘍浮腫や炎症の存在を得ることができることです。研究者に。

<table border="1"><tbody><tr><td> 試薬や供給の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> GL261 - luc2バイオウェアウルトラ</td><td>キャリパーライフサイエンス</td><td> GL261 - luc2 </td><td></td></tr><tr><td>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> 10313039 </td><td></td></tr><tr><td>ジェネティシン（G418） </td><td> GIBCO（インビトロジェン） </td><td> 11811-023 </td><td></td></tr><tr><td>ウシ胎児血清（FCS） </td><td>インビトロジェン</td><td> 26140079 </td><td></td></tr><tr><td> Phospateは緩衝生理食塩水（PBS） </td><td>インビトロジェン</td><td> 70011044 </td><td></td></tr><tr><td> C57BL/6-cBrd/cBrd/Crマウス</td><td> NCI -フレデリック</td><td></td><td></td></tr><tr><td>アクワの涙の潤滑眼軟膏</td><td> Akorn株式会社</td><td> 17478-062-35 </td><td></td></tr><tr><td> Ketaset（塩酸ケタミン） </td><td>ワイエス</td><td> 11570775 </td><td></td></tr><tr><td> Sedazine（キシラジン塩酸塩） </td><td>ワイエス</td><td> 10031894 </td><td></td></tr><tr><td>小動物の定位音源</td><td> Kopfは楽器</td><td> 900 </td><td></td></tr><tr><td> SYS - Micro4コントローラとUltraMicroPump </td><td>世界の精密機器</td><td> UMP3 - 1 </td><td>利用できない場合は、手動で注入することが可能です</td></tr><tr><td> 26ゲージと10μlのシリンジは、針の面取り</td><td>世界の精密機器</td><td> SGE010RNS </td><td></td></tr><tr><td> Adison鉗子</td><td>世界の精密機器</td><td> 500092 </td><td></td></tr><tr><td>ペンフィールド解剖</td><td>タラ漁船</td><td> 65から1015 </td><td></td></tr><tr><td> 16グラム1 ½精密グライドニードル</td><td> Beckton、ディッキンソン社（BD） </td><td> 305198 </td><td></td></tr><tr><td>外科ブレードハンドル</td><td> BD </td><td> 371030 </td><td></td></tr><tr><td>サイズ15ブレード</td><td> BD </td><td> 371315 </td><td></td></tr><tr><td> 4から0 Vicryl縫合糸</td><td>エチコン</td><td> VCP496G </td><td></td></tr><tr><td>骨ろう</td><td> MEDLINE </td><td> DYNJBW25 </td><td></td></tr><tr><td> Povidine -ヨウ素綿棒スティック</td><td> MEDLINE </td><td> MD93901 </td><td></td></tr><tr><td> D -ルシフェリンカリウム塩</td><td>キャリパーライフサイエンス</td><td> 122796 </td><td></td></tr><tr><td> Forane（イソフルラン） </td><td>バクスター</td><td> 1001936060 </td><td></td></tr><tr><td> OPMI Pentero顕微鏡</td><td>カールツァイス株式会社</td><td></td><td>どんな手術用顕微鏡で十分です</td></tr><tr><td>オプションの麻酔システムとXenogen IVISスペクトラム</td><td>キャリパーライフサイエンス</td><td></td><td></td></tr></tbody></table>

intracranial implantation with subsequent 3d in vivo bioluminescent imaging of murine gliomas

マウス神経膠腫261（GL261）はin vivoでのモデル系として認識されているヒトの多形性膠芽腫（GBM）の機能の多くは反復する。細胞株は、もともとC57BL / 6同系マウスの系統1に3 -メチル- cholantreneの頭蓋内注射によって誘発されたので、免疫学的に有能なC57BL / 6マウスを使用することができます。我々はGL261を使用していますが、次のプロトコルは、任意の頭蓋内のマウス腫瘍モデルの移植とモニタリングに使用することができます。 GL261細胞を安定的に発現するホタルルシフェラーゼ（GL261 - LUC）に設計された。我々はまた、CMVプロモーターから発現luc2遺伝子の安定なトランスフェクションにより、明るくGL261 - luc2細胞株を作成した。国立癌研究所、フレデリック、MDからC57BL/6-cBrd/cBrd/Crマウス（C57BL / 6のアルビノ変異体）は黒の皮と毛皮による光の減衰を除去するために使用されていました。 in vivoイメージング生体 IM で IVISのスペクトラムを使用して、アルビノC57BL / 6マウスを使用するとシステムの老朽化と、移植の日（キャリパーライフサイエンス、ホプキントン、MA）から可能です。 GL261 - lucおよびGL261 - luc2細胞株は、親のGL261細胞として生体内挙動に同じことを示した。人間性GBMに存在する共有組織学的特徴とこのマウスモデルのようなものがあります：腫瘍壊死、pseudopalisades、血管新生、浸潤、細胞過多、および炎症1。 移植前の動物には、以上のメスで作成された定位装置や切開に入れ、ケタミン（50 mg / kgの）、キシラジン（5 mg / kg）をし、ブプレノルフィン（0.05 mg / kg体重）の腹腔内注射により麻酔し、頭蓋正中線。 burrholeはブレグマ後方0.1ミリメートルと正中線の右側に2.3ミリメートルとした。針は2.6mmの深さに3mmから撤退0.4mmの深さに挿入した。 GL261リュックまたはGL261 - luc2細胞（10 7細胞/ ml）の二つの液を3分間かけて注入した。 burrholeが閉じられましたbonewaxと切開を縫合したと。 定位の移植後の生物の細胞は、移植の日から検出され、腫瘍は、IVISのスペクトラム測定器の3次元画像再構成機能を使用して分析することができます。動物は、150μgルシフェリン/イメージングの前に体重20分の皮下注射を受ける。全身腫瘍組織量が時間の経過とともに平均腫瘍の生物発光を用いて定量される。担癌マウスは、罹患率を評価するために、毎日観察し、以下のいずれかまたは複数の現象が存在するときに安楽死さ：無気力、歩き回るに失敗、猫背の姿勢、新郎への障害、食欲不振、体重の&gt; 10％が失われる。腫瘍は剖検上の動物のすべてに明白であった。

Watch this Scientific Journal Video about Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas at JoVE.com

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

C57BL / 6マウスにGL261細胞の頭蓋内注入は、人間の多形性膠芽腫の特徴の多くを再現悪性神経膠腫を生成します。私達は私達が使用できるようにするために安定的にルシフェラーゼを発現するGL261細胞を用い<em&gt; in vivoで</em&gt;イメージングは​​腫瘍の進行に従うこと。手術と3D<em&gt; in vivoで</em&gt;画像が実証されています。

その後の3Dと頭蓋内注入インビボでの</emマウス神経膠腫の>生物発光イメージング

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

intracranial-implantation-with-subsequent-3d-vivo-bioluminescent

Research

JoVE Journal

Medicine

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

20.3K ビュー.  Barrow Neurological Institute of St. Joseph’s Hospital and Medical Center. 中文

ビデオ: Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

マウス生物発光肝腫瘤モデル

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

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 ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

マウスでのせん断応力誘発アテローム性動脈硬化症をトリガするための頸動脈カフの注入

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

定位頭蓋内注入と<emインビボ&gt;</em多形性膠芽腫のマウスモデル系における腫瘍異種移植片の&gt;生物発光イメージング

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

再狭窄の研究のための頸動脈内ステント留置のマウスモデル

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

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

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

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

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

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

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

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

マウス内視鏡検査のため<em&gt;インビボ</em&gt;発がんと腸の評価のマルチモーダルイメージング創傷治癒および炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

血管周囲ドラッグデリバリーパッチの移植に大腿動脈ワイヤ傷害のマウスモデル

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

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 ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

光シート顕微鏡による劇症心筋炎のマウスモデルにおける白血球浸潤の定量的可視化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

その後の3Dと頭蓋内注入インビボでの生物発光イメージング