以下のプロトコルは、20〜35グラムのマウスではうまく機能システムが記載されています。この方法は、容易に、単に、カテーテルのサイズを変更することによって、より大きな又はより小さなマウスに適応することができました。全ての動物プロトコルは、ジョンズホプキンス大学動物実験委員会によって承認されました。 1.肺点滴注入<ol><li>挿管に使用する商業1インチ長い20グラム静脈カニューレを選択します。 </li><li>手動で、図1に示すように、それは先端のわずかな曲率を生成するために、曲げカテーテル先端を変更します。 </li><li>ケタミンの混合物（100mg / kg）およびキシラジン（15 mgの/ kg）でマウスを麻酔IPを注入し、反射運動の欠如によって麻酔を確認します。すぐに麻酔した後、目に獣医軟膏を適用します。麻酔が目に獣医軟膏を適用し、手術後や点滴鎮痛のためにカルプロフェン（5-10ミリグラム/キログラムSQ）を得た直後。 </li><li>畝を配置傾斜プラットフォーム上でSE仰向け。 図2に示すように 、縫合糸ループを有する大規模オフィスバインダーは完璧に作品をテープで固定しました。 </li><li>首とクリーンの腹の部分を剃るし、70％アルコールで首エリアを消毒。新しいlatex-とpowder-無料の手袋を使用すると、70％アルコールで消毒手術器具を使用しています。 </li><li>鋭いハサミを使用すると、約12ミリメートル下顎切歯の下に首に小さな外科的切開を行います。 </li><li>気管の腹側の壁を見ることができるまで、ピンセットで軽く首尾側で皮膚を引っ張ります。 </li><li>そっと舌を撤回し、マウスの腹面に向かって傾斜して曲がった先端でカニューレを挿入します。 1.4のように、首の皮膚を軽く引いて、気管にカニューレを挿入します。 注：少し練習すると、カテーテルが気管を下に移動する表示されます。それが食道に入る場合には、カテーテルの動きの視覚的な目撃情報はありません。いいえ切開ん気管内に作られています。 </li><li>カテーテルを首の気管に見られると、約5mm、確実に声帯を通過したが、まだ十分に気管分岐部の上にすることを進みました。 </li><li>ゲルローディングピペットチップでカテーテルを介して注入することにより、液体の50μlにまで浸透させるための準備。ルアーハブでチップを置きますが、マウスの呼吸で先端同期内の流体の動きを観察するために慎重に見て注入する前に。その後、点滴を注入。 </li><li> 1mlシリンジを使用すると、すぐに肺の中の液体の深いを分散を助けるためにカテーテルを介して肺に空気の0.6ミリリットルの比較的急速なインフレを行います。カニューレを削除します。 </li><li>カニューレを削除します。 </li><li> VetBondための添付文書の指示通りに小さな手術創を閉鎖するためにシアノアクリレート接着剤の少量を使用してください。個々のケージにマウスを置き、彼らが目を覚ますまで、視覚的に監視し、discomfの兆候なしで正常に動作ORT。 </li></ol> 2.肺固定注：すべての実験手順は、マウスで行われると、肺がホルムアルデヒド（または任意の他の所望の固定剤）で固定することにより組織学的処理のために準備することができます。 <ol><li> IACUC許容可能な手順でマウスを生け贄に捧げます。ビデオに示されている代表的なマウスの場合、麻酔したマウスの頚椎脱臼が使用されます。 </li><li>外科、気管の腹側を露出させ、小さなカットを作る、と気管に18 Gスタブ針の先端を挿入し、スレッドとそれを結びつけることによって（まだ行っていない場合）気管切開を行います。 </li><li>慎重に、正中切開で胸郭を開き、ダイヤフラムを切り取って、肺を露出するために胸部側面の壁を取り除きます。 </li><li>ホルムアルデヒドを含むリングスタンド上の容器に針のルアー端を接続します。 図3を参照してください 。 </li><li> 25センチメートルMOUのレベルを超えてホルムアルデヒドの上面を設定SE。 図3を参照してください 。次は、コックの終わりから流体を実行して、固定チューブ内に空気が存在しないことを確認してください。貯留管に気管カニューレのルアー端を接続します。ホルムアルデヒドで肺を膨らませるコックを開きます。少なくとも20分間、圧力下肺を残します。 </li><li>ホルムアルデヒドで肺を膨らませるコックを開きます。少なくとも20分間、圧力下肺を残します。 </li><li>次に、スタブ針の端部を越えて気管を縛ります。それはuncannulated気管の多くを露出するために、針の上にゆっくりと引き戻すのに役立つことがあります。安全に接続すると、ストップコックを削除します。 </li><li>慎重に肺を解剖します。 </li><li>一晩ホルムアルデヒドで肺を置きます。長い時間が微細であり、シミや手順が特定の時間を指定することができます。また、z軸を修正などの他の液体の固定剤は、点滴注入、浸漬のために使用することができます。 </li><li>さらに組織学的処理の前に、測定次に説明するように固定された肺容量。 </li></ol>固定肺容量の3測定<ol><li> 図4に示すように、アルキメデスの原理を用いて、肺容量を測定します。ホルムアルデヒドから固定肺を外し、心臓や他の非肺組織を分析。 </li><li>完全に水の下で肺を維持するために使用される以前に構築シンプルな自家製のワイヤ支持デバイスを使用してください。 注：このデバイスが使用されている方のバランスと両立させることが必要です。 図5に示される典型的な装置は、プラスチックピペットと薄い（20 G）のワイヤから形成されています。このシステムは、ビデオで使用さバランスでうまく動作しますが、簡単にほとんどの研究室残高に適合させることができます。 </li><li>水の代わりに、サポートするケージとのバランスと風袋の水の≈200mlのビーカーを置きます。 図6を参照してください金属ケージを取り外します。水面に肺を置き、下に押して、水ケージと。 </li><li>バランスの重量を記録します。この数は、置換された水の量を反映しているため、肺容量の直接の尺度です。肺または縫合糸またはワイヤケージのいずれかの部分が側面やビーカーの底に接触しないことを確認してくださいするように注意してください。 </li><li>精度については、この測定を繰り返します。水から肺を取り出し、組織の乾燥。再び所定の位置にケージを入れたビーカーを風袋引きし、肺容量測定を繰り返します。 2つのボリュームの測定値は、その後、平均化されるべきです。 注：肺がより約一週間のためにホルマリン中に残っている場合は、肺の中の空気が液体に溶解されます。この場合、肺は沈むので、水没肺を維持するために、図5のような任意のデバイスを使用する必要がなくなります。このような場合、ボリュームが、図4に示されるように、それが完全に浸漬されるまで、縫合糸のいずれかの文字列によって肺を保持することによって簡単にすることによって測定することができます。 </li></ol>

<ol>
	<li>Brown, R. H., Walters, D. M., Greenberg, R. S., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+endotracheal+intubation+and+pulmonary+functional+assessment+for+repeated+studies+in+mice.">A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice.</a> J Appl Physiol. 87, 2362-2365 (1999).</li><li>Walters, D. M., Wills-Karp, M., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cellular+profile+and+lung+function+with+repeated+bronchoalveolar+lavage+in+individual+mice.">Assessment of cellular profile and lung function with repeated bronchoalveolar lavage in individual mice.</a> Physiol Genomics. 2, 29-36 (2000).</li><li>Hamacher, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+wire+guide-based+orotracheal+mouse+intubation:+description,+evaluation+and+comparison+with+transillumination.">Microscopic wire guide-based orotracheal mouse intubation: description, evaluation and comparison with transillumination.</a> Laboratory animals. 42, 222-230 (2008).</li><li>Spoelstra, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+simple+method+for+endotracheal+intubation+of+mice.">A novel and simple method for endotracheal intubation of mice.</a> Laboratory. 41, 128-135 (2007).</li><li>Zhao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+retrograde+intubation+in+mice.">A technique for retrograde intubation in mice.</a> Lab animal. 35, 39-42 (2006).</li><li>Rivera, B., Miller, S., Brown, E., Price, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+endotracheal+intubation+of+mice+and+rats+used+in+imaging+studies.">A novel method for endotracheal intubation of mice and rats used in imaging studies.</a> Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science. 44, 52-55 (2005).</li><li>Vergari, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+orotracheal+intubation+in+mice.">A new method of orotracheal intubation in mice.</a> European review for medical and pharmacological sciences. 8, 103-106 (2004).</li><li>Vergari, A., Polito, A., Musumeci, M., Palazzesi, S., Marano, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Video-assisted+orotracheal+intubation+in+mice.">Video-assisted orotracheal intubation in mice.</a> Laboratory animals. 37, 204-206 (2003).</li><li>Hastings, R. H., Summers-Torres, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Laryngoscopy+in+Mice.">Direct Laryngoscopy in Mice.</a> Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science. 38, 33-35 (1999).</li><li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.</a> Am J Respir Crit Care Med. 181, 394-418 (2010).</li><li>Foster, W. M., Walters, D. M., Longphre, M., Macri, K., Miller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+the+measurement+of+mucociliary+function+in+the+mouse+by+scintigraphy.">Methodology for the measurement of mucociliary function in the mouse by scintigraphy.</a> J Appl Physiol. 90, 1111-1117 (2001).</li><li>Cai, Y., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+intratracheal+intubation+to+study+the+pathology+and+physiology+of+mouse+lung.">Noninvasive intratracheal intubation to study the pathology and physiology of mouse lung.</a> Journal of visualized experiments : JoVE. , e50601 (2013).</li><li>Das, S., Macdonald, K., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+mouse+lung+intubation.">A simple method of mouse lung intubation.</a> Journal of visualized experiments : JoVE. , (2013).</li><li>MacDonald, K. D., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+simple+method+of+mouse+lung+intubation.">An improved simple method of mouse lung intubation.</a> J Appl Physiol. 106, 984-987 (2009).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+10%+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage.</a> American Reveiw of Respiratory Diseases. 132, 1078-1083 (1985).</li><li>Bishai, J., Fields, M. J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Mouse+Lung+Volumes+Inflated+with+Air+and+Instilled+Fixatives.">Comparison of Mouse Lung Volumes Inflated with Air and Instilled Fixatives.</a> Proc Am Thorac Soc. 3, A549 (2006).</li><li>Mitzner, W., Brown, R., Lee, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+lung+volumes+in+mice.">In vivo measurement of lung volumes in mice.</a> Physiol Genomics. 4, 215-221 (2001).</li></ol>

ここで説明する手順は、いくつかの利点を有します。まず必要な機器は、簡単で安価です。第二に、挿管はすぐにいくつかのエラーで行うことができます。第三に、固定された肺容量を一定の圧力で肺を固定した後、測定する能力は、肺10内の構造や細胞の適切な定量化を可能にします。 挿管に対する1つの可能な欠点は、簡単な手術です。これは、2 番目の</...

多くの理由により、肺癌は、マウスにおいて広く研究されていません。その理由の一つは、肺へのアクセスは、インビボで非常に困難であり、固定肺の定量分析は、一般的に行われないということです。このホワイトペーパーで説明する方法は、この状況を改善するように設計されています。目標は、本明細書に大きく肺癌または他の病理との設定、マウスの肺の分析を補助する単純な方法を記載することです。これらのアプローチのいずれも、全く新しいませんが、彼らはここで説明するように簡略化して、スタンドアロンの方法として一緒に提示されていません。 主に縦方向の研究における個々のマウスで繰り返し肺機能や気管支肺胞洗浄を行うことを目的に、マウスの肺の挿管のための方法を記載している原稿の数がありました。そのオリジナルの論文以来、覚書に異なるアプローチが記載されている他のいくつかの論文がありましたE挿管1 -9。これらの方法の全てが正常に使用することができるが、それらは通常、かなりの訓練を必要とし、重要な故障率なしに、多くの場合ではありません。加えて、肺機能測定を行うために、カニューレには、空気漏れがないようにしっかりと十分な気管にフィットする必要があります。しかし、挿管のための別の実用化は、肺に直接特定の薬剤（癌細胞または他の傷害）、または治療薬を送達することです。このような手順は、タイトなフィッティングカニューレも任意の洗練された肺機能の機器を必要としません。ここに示されているこの方法の新規な特徴は、食道に入るカニューレのいずれかの可能性なしに挿管を可能にする簡単な外科的処置を必要とします。この単純なアプローチは比較的少ないトレーニングや経験を持つ成功した挿管を可能にします。以下のような多くの30匹のマウス/時間がゼロに近づく故障率と、この方法を用いて治療することができます。 Tたら彼のマウスは、負傷または癌性肺が、その後組織学的および病理学的分析のために取り外すことができ、犠牲にする準備ができています。しかし、適切に他の肺との比較のために任意の組織学的変数を定量化するためには、固定手順を標準化し、適切に固定された肺容積10を定量化することが不可欠です。本稿では詳細に標準化された固定手順と同様に固定肺容量を測定する方法を可能にする簡単な手順について説明します。そのようなボリューム決意することなく、唯一の相対密度が10を測定することができるので、体積は、組織学の定量化に不可欠な測定基準です。肺気量がわかれば、しかしながら、肺の細胞、および他の構造の測定値の絶対的な測定値は、その後、定量することができます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Laboratory Balance</td>
			<td>Ohaus</td>
			<td>Adventurer Pro Model AV 313&#160;</td>
			<td>Other balances can be used if they have a range of 1 - 300g</td>
		</tr>
		<tr>
			<td>20 g Luer intravenous catheter</td>
			<td>Insylte</td>
			<td></td>
			<td>Several other possible vendors, e.g., Jelco Optiva</td>
		</tr>
		<tr>
			<td>500 mL laboratory bottle</td>
			<td>Various</td>
			<td></td>
			<td>Several other possible vendors</td>
		</tr>
	</tbody>
</table>

instillation and fixation methods useful in mouse lung cancer research

負傷またはマウスを殺すことなく、直接肺に生き薬、細胞、または化学物質を植え付けるする能力は、肺癌研究における重要なツールです。肺機能の測定のためにマウスを挿管する方法を示す公開されている多数の方法がありますが、どれもマウスの大規模コホートにおける急速な気管点滴注入のための潜在的な問題もなくはありません。本研究では、簡単かつ迅速な方法は、効率的な方法でそのような点滴注入を実施する研究者を可能にすることが記載されています。この方法は、特殊な工具や照明を必要とせず、非常に少し練習して学ぶことができます。これは、マウスを麻酔気管を可視化するために、首に小さな切開し、その後、直接静脈カテーテルを挿入することを含みます。小さな切開を迅速に組織接着剤で閉じ、マウスを回復させます。熟練した学生や技術者が2分/マウスの平均速度で点滴を行うことができます。 Tたら癌が確立されている彼、肺の定量的な組織学的分析の必要性がしばしばあります。伝統的に病理学者は、通常、固定時に肺膨張を標準化するために気にしない、との分析は、多くの場合、非常に主観的スコアリングシステムに基づいています。これはいつか肺腫瘍の大きさの粗推定値のために十分に適切であり得るが、肺の構造または細胞の任意の適切な立体定量化は、再現固定手順とそれに続く肺容量の測定を必要とします。ここでは、両方が圧力下で肺を固定して、正確に固定された肺容量を測定するための簡単​​な信頼性のある手順について説明します。唯一の要件は、1 MG-300グラムの範囲に亘って正確である実験室のバランスです。ので、ここで提示された手順が大幅にマウスで肺癌を作成、治療、および分析する能力を向上させることができます。

手順は、それ自体で任意の一般的な結果にはならない最初のプロトコルで説明します。それが唯一の直接気管内に物質を植え付けるために非常に信頼性の高い手段が記載されている。 図7は、トリパンブルーは、ここで説明する方法で点眼した肺の一例を示しています。気管またはマウス11,12に直接与えられた他の染料またはトレーサーで見てきたものに類似した色素の?...

Watch this Scientific Journal Video about Instillation and Fixation Methods Useful in Mouse Lung Cancer Research at JoVE.com

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

本稿の目的は、大幅に肺癌または他の病状とのセットアップとマウスの肺の分析を支援する単純な方法を記述することです。我々は、簡単かつ確実に肺の点滴注入、固定、および肺容量の測定を行うために3プロトコルを提示します。

マウス肺癌研究における有用な点滴や固定方法

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

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JoVE Journal

Medicine

28.1K ビュー.  Johns Hopkins University. 本稿の目的は、大幅に肺癌または他の病状とのセットアップとマウスの肺の分析を支援する単純な方法を記述することです。我々は、簡単かつ確実に肺の点滴注入、固定、および肺容量の測定を行うために3プロトコルを提示します。 

ビデオ: Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

医学

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

マウス肺挿管の簡単な方法

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

マウス肺の病理生理学を研究するための非侵襲的気管内挿管

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

マウス絞り機能の診断用超音波イメージング

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

This article describes bronchoscopic techniques in the bovine lung under experimental conditions, i.e. bronchoscopically guided inoculation, bronchoalveolar lavage, bronchial brushing, and transbronchial lung biopsy.

生物医学研究における牛の肺：視覚ガイド付き気管支鏡検査、気管支内接種および<em&gt;インビボ</em&gt;サンプリングテクニック

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

挿管媒介気管内（IMIT）点眼：非侵襲的、肺特異的送達システム

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

トランスレーショナルがん研究のためのヒト組織での作業

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

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

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

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

定量的リアルタイムPCRによるマウス乳腺腫瘍モデルにおける肺転移の評価

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

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.