Name: perfusion and inflation of the mouse lung for tumor histology
Uploaded: 2020-08-06T15:30:00.000+00:00
Duration: 4 min 24 s
Description: Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

本論文では、国立トランスレーショナルサイエンス推進センターが、賞番号UL1TR003096(MDE)、国立心臓、肺、血液研究所肺疾患研修プログラム5T32HL134640(MLD)の下で支援を受けました。

ここに記載されているすべての方法は、バーミンガムのアラバマ大学の制度的動物ケアと使用委員会(IACUC)によって承認されています。
1. 実験プロトコル
<ol><li>承認された IACUC 方式を使用してマウスを犠牲にします。ここでは、5%イオブルランで麻酔したマウスの子宮頸部脱臼を用いた。研究に適切なマウスを使用します。ここでは、8週齢のFVBマウスを使用しています</li>
	<li>手術用ハサミを使用して、下腹部の真ん中に3.5〜5mmの水平切開を行います。次に、切開から作成された小さな穴に外科用ハサミを挿入し、マウスの首のすぐ下に中央の正中線を垂直に切断します。</li>
	<li>指で皮膚を引き戻し、腋窩リンパ節を検査します。</li>
	<li>手術用ハサミを使用して、3.5mmの側面切開を行い、腹腔を開き、胸部の底まで前方向に切断します。腹腔内の臓器を検査します:肝臓、脾臓、腎臓など。</li>
	<li>手術用ハサミの平らで、または鉗子を使用して、肝臓を動かして横隔膜を露出させる。横隔膜の腫瘍の成長や転移を検査します。その後、オペレータの右側にあるダイヤフラムをそっと切り取り、拡大します。横隔膜を右から左にそっと切り、胸腔と肺を露出させる。肺を切らないように注意してください。</li>
	<li>左肋骨ケージの横端(オペレータの右)を切り取って、肺の左葉を検査します。</li>
	<li>肺の右葉をそっと動かし、右肋骨ケージの横端を切り取り、リブケージを取り除きます。 注:リブケージの除去はオプションですが、除去は後の肺インフレのより明確なビューを可能にします。<ol>
<li>新鮮な肺組織または凍結した肺組織が必要な場合は、ヘモスタット鉗子を使用して左葉の気管支をクランプし、灌流前に外科用ハサミを使用して左肺を切除する。</li>
	</ol>
</li>
	<li>鉗子を使用して気管を覆う組織を持ち上げ、余分な組織を切り取る。その後、気管を裏打ちする薄い組織をそっと切り、気道を露出させる。</li>
	<li>外科用はさみで腎動脈を切り抜ける。</li>
	<li>肺を浸透させるために、22G針の3 mLシリンジを使用して、心臓の右心室に10 U/mLヘパリンを含む1x PBSを注入する。PBS/ヘパリンで約300μL/秒でゆっくりと肺を透過します。肺は頻繁に白くなります。このステップでは、通常、2.5 mL の PBS が使用されます。</li>
	<li>肺膨張のために、22G針を用いた3mLシリンジを使用し、今度は気管に平行に保持した。針を気管に入れ、肺が完全に膨張するまで流量が〜200μL/s以下の10%ホルマリンを注入します。肺が膨張すると、ホルマリンは気管から逆流します。針を数秒間所定の位置に保持し、引き出します。<ol>
<li>(オプション)針と肺の膨張を引き出す前に、縫合糸を使って気管を締め出してください。これを達成するには、小さな鉗子で糸のポイントを保持する縫合糸の4インチを使用してください。気管の裏側に糸を置き、引き抜いて針の周りにループを作ります。次に針の周りにオーバーハンドの結び目を作ります。結び目をしっかりと引っ張り、気管から針を取り除き、結び目を閉じます。</li>
	</ol>
</li>
	<li>鉗子を使って心臓を持ち上げ、肺の真後ろに外科用のはさみを挿入し、心臓を持ち上げて肺を切除しながら結合組織を切断する。</li>
	<li>肺からそれを取り除くために心臓をカットします。</li>
	<li>肺にマウスIDまたは研究IDでラベル付けされたカセットを置き、カセットを10%緩衝ホルマリンに入れ、24〜48時間固定します。肺は、必要に応じて1年以上固定剤に残すことができます。</li>
	<li>肺を含むカセットを70%エタノールに移し、占いの処理に進みます。</li>
</ol>

<ol>
	<li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R., Structure, A. <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> American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).</li><li>Vasilescu, D. M., Knudsen, L., Ochs, M., Weibel, E. R., Hoffman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+murine+lung+preparation+for+detailed+structural+evaluation+via+micro-computed+tomography.">Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography.</a> Journal of Applied Physiology. 112 (1), 159-166 (2012).</li><li>Blumler, P., Acosta, R. H., Thomas-Semm, A., Reuss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fixation+for+the+preservation+of+air+spaces.">Lung fixation for the preservation of air spaces.</a> Experimental Lung Research. 30 (1), 73-82 (2004).</li><li>Oldmixon, E. H., Suzuki, S., Butler, J. P., Hoppin, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+dehydration+fixes+elastin+and+preserves+lung+air-space+dimensions.">Perfusion dehydration fixes elastin and preserves lung air-space dimensions.</a> Journal of Applied Physiology. 58 (1), 105-113 (1985).</li><li>Braber, S., Verheijden, K. A., Henricks, P. A., Kraneveld, A. D., Folkerts, 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+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema.">A comparison of fixation methods on lung morphology in a murine model of emphysema.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 299 (6), 843-851 (2010).</li><li>Renne, R., 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=Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies.">Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies.</a> Toxicologic Pathology. 29 (5), 587-589 (2001).</li><li>Limjunyawong, N., Mock, 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=Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research.">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> Journal of Visualized Experiments. (102), e52964 (2015).</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.+A+comparison+of+eleven+laboratory+species.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage. A comparison of eleven laboratory species.</a> American Review of Respiratory Disease. 132 (5), 1078-1083 (1985).</li><li>Edmonds, M. D., 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=MicroRNA-31+initiates+lung+tumorigenesis+and+promotes+mutant+KRAS-driven+lung+cancer.">MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer.</a> Journal of Clinical Investigation. 126 (1), 349-364 (2016).</li><li>Zhao, K., 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=Wogonin+suppresses+melanoma+cell+B16-F10+invasion+and+migration+by+inhibiting+Ras-medicated+pathways.">Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.</a> PLoS One. 9 (9), 106458 (2014).</li></ol>

著者らは開示するものは何もない。

マウス肺の灌流、膨張、および固定のために上記の手順は、肺腫瘍組織学および病理解析のためのマウス肺の迅速かつ効率的な調製に最適である。この手順は特別な装置を必要とせず、マウス1個につき6分以内に実行できます。この手順では、インフレや一定の流体圧力に固定体積は必要ありません。この手順は標準化されていないため、肺の立体学的または形態測定を行うことを望む人に?...

肺腫瘍発生や癌転移から肺への様々なマウスモデルは、複雑なGEMMから発がん性および異種移植片モデルに至るまで、癌細胞が心臓内、胸腔内、尾静脈、または肺内に腫瘍を確立する他の方法を介して注入される同種および異種移植片モデルに至るまで存在する。これらのモデルはすべて、肺組織学と病理の組織学的評価に共通する必要性を共有しています。したがって、余分な血液を除去するために肺を浸透させ、肺を膨らませて肺のアーキテクチャを明確に可視化しながら、マウスの壊死を行う堅牢かつ迅速な方法が必要である。速度は、単一の時点で数十匹のマウスから肺を収集する必要があるため、この手順の重要な要素です。この手順は、マウスあたり6分以内に実行できます。
この手順は腫瘍組織学を評価するのに十分ですが、肺のステテロロジーまたは形態測定を行いたい人には推奨されません。このような測定では、肺の絶対表面積、絶対体積、歯槽サイズおよび番号1の計算と同様に、肺の膨張を標準化する必要があります。この方法は、一部のイメージングアプローチにも最適ではありません。例えば、ex vivoモルフォメトリック解析のためにμCTを介して肺をイメージングするには、肺が空気2で満たされたままである必要があります。空気空間および寸法の保存が第一の関心事である場合、過灌流脱水技術3、4によって肺を固定することが推奨される。このモデルの最大の懸念の1つは、肺胞壁の破裂の可能性であり、肺気腫の研究における使用を減らす。しかし、肺気腫の研究のための肺の固定のための推奨手順は、一定の流体圧力下で(ここで説明するプロトコルに類似した)10%ホルマリンの気管内点眼点眼によって、またはその場で固定5で肺を固定することが推奨されるように、まだ非常に似ています。
ここで説明した手順の利点は、一定の流体圧力を必要とせず、肺が完全に膨張するまで膨張し、処置に必要な時間を短縮することです。ここで説明する手順は、毒物学病理学会の軍備間膜によって推奨される方法によく似ており、毒物学研究のための肺固定の最良の方法を推奨する小委員会が結成された。この小委員会内の科学者の大半は、肺が固定剤6に残された時間に様々な勧告があったが、注射器で気管内点眼によって肺を固定することを推奨した。従って、肺の膨張および固定の様々な方法が存在する一方で、本明細書に記載される方法は、下流腫瘍組織学的評価のために肺を迅速に膨張および固定する最適な方法であると提案される。

<table><tbody><tr><td>10% buffered formalin</td><td>Fisher</td><td>23-245685</td><td></td></tr><tr><td>22 G Needle</td><td>BD</td><td>305155</td><td></td></tr><tr><td>3 mL syringe</td><td>BD</td><td>309656</td><td></td></tr><tr><td>70% Ethanol</td><td>Decon</td><td>2405</td><td></td></tr><tr><td>Forceps</td><td>Harvard Apparatus</td><td>72-8595</td><td></td></tr><tr><td>Heparin</td><td>Fisher</td><td>H19</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>Surgical scissors</td><td>Harvard Apparatus</td><td>72-8428</td><td></td></tr></tbody></table>

perfusion and inflation of the mouse lung for tumor histology

肺の病態を評価する能力は、肺がん研究やがん転移の分野で重要です。調達した組織の質を犠牲にすることなく、研究から迅速かつ効率的に壊死を行うことも同様に重要です。このプロトコルの目的は、下流の組織学的分析のためにマウス肺を迅速に穿ファズ、膨張、および固定する方法を提示することです。この方法は、肺の膨張を標準化しません。したがって、特別な手順や機器を必要とせず、心臓を通して灌流した後の気管を通して直接固定液を植え付けるだけです。これにより、腫瘍のサイズ、組織学、およびスコアリングの十分な推定が可能になります。これはまた肺組織の固定の前に凍結組織のコレクションを可能にする。この方法は、後で肺の形態学的定量を可能にしないために制限されています。しかし、遺伝子組み換えマウスモデル(GEMM)、異種モデル、異種移植片腫瘍および転移研究による肺腫瘍分析には十分です。

上記のプロトコルはマウス肺の速い潅流、膨張および固定を可能にする。以下の図は、各ステップの重要性を表しています。 図1 は、灌流ステップがスキップされたか、肺が正しく穿フューする失敗したPBSおよび肺と浸透したH&E染色された肺を示している。示されているように、不十分な浸透性肺の過剰な血液は理想的な構造学よりも少なく、肺アーキテクチャを完全?...

Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

Perfusion and Inflation of the Mouse Lung for Tumor Histology

この方法の目的は、肺腫瘍病理の検査および肺への転移の評価のためのマウス肺の灌流、膨張、および固定のための簡単で効率的な方法を提示することである。

腫瘍組織学におけるマウス肺の多灌流とインフレーション

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

perfusion-and-inflation-of-the-mouse-lung-for-tumor-histology

Research

JoVE Journal

Cancer Research

18.2K ビュー.  University of Alabama at Birmingham. この方法の目的は、肺腫瘍病理の検査および肺への転移の評価のためのマウス肺の灌流、膨張、および固定のための簡単で効率的な方法を提示することである。

ビデオ: Perfusion and Inflation of the Mouse Lung for Tumor Histology

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

真空安定化撮像窓と肺における長期高分解能生体顕微鏡

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

がん研究

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

肺癌のマウスモデルにおける糖代謝の動的変化を測定する18F-FDG PET/CT イメージングと定量組織学を活用

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

組み合わせによるマウスのシングルセル解像度で肺転移の可視化の向上<em&gt;のin-situ</em肺組織とのX-gal染色の&gt;灌流<em&gt; lacZを</em&gt;タグ付き腫瘍細胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

医学

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

マウス肺切除術および義歯移植による内臓肺表面積の標準化法

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

発生生物学

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

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 research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

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 ...

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

同所性同種肺がん細胞生着の効率を高めるブレオマイシンとマウスの気道をプレコンディショニング

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.

Presented is a method for air-inflation with vascular perfusion-fixation of the lungs that preserves the location of cells within airways, alveoli and interstitium for structure-function analyses. Constant airway pressure is maintained with an air-inflation chamber while fixative is perfused via the right ventricle. Lungs are processed for histologic studies.

血管灌流固定を用いたマウス肺の空気膨張

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, ...

神経科学

<meta charset="utf8"></head><body>マウス肺移植における細胞動態のリアルタイムモニタリング

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>著者スポットライト:Intravital Imagingによる肺移植免疫学の進歩

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

移植されたマウス肺の生体内2光子顕微鏡検査

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

腫瘍組織学におけるマウス肺の多灌流とインフレーション

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組み合わせによるマウスのシングルセル解像度で肺転移の可視化の向上

マウス肺切除術および義歯移植による内臓肺表面積の標準化法

マウス肺癌研究における有用な点滴や固定方法

同所性同種肺がん細胞生着の効率を高めるブレオマイシンとマウスの気道をプレコンディショニング

血管灌流固定を用いたマウス肺の空気膨張

移植されたマウス肺の生体内2光子顕微鏡検査

移植されたマウス肺の生体内2光子顕微鏡検査

移植されたマウス肺の生体内2光子顕微鏡検査