Name: automated measurement of pulmonary emphysema and small airway remodeling in cigarette smoke-exposed mice
Uploaded: 2015-01-16T14:00:00.000+00:00
Duration: 10 min 37 s
Description: Watch this Scientific Journal Video about Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice at JoVE.com

私たちは、フランチェスカPolverino MD、この記事への彼女の貢献のためのブリガム·アンド·ウィメンズ病院での研究員、そしてまたモニカ八尾、BS、およびケイト·ライデル、ネズミ畜産とタバコの煙にマウスを曝露との援助のためにBSに感謝したい。 この作品は、公衆衛生局、国立心臓、肺、血液研究所補助金HL111835、HL105339、HL114501、客室乗務員医学研究所助成＃CIA123046、ブリガム·アンド·ウィメンズ病院·ラブレース呼吸器研究所コンソーシアム、ケンブリッジNIHR医学によってサポートされていました研究センター。

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We have no conflicts of interest to disclose.

Most researcher use mice to model the main chronic lung pathologies and abnormal lung physiology in COPD (airspace enlargement, SAR, and increases in lung compliance) present in the human disease. A comprehensive approach to assess the effect of molecules of interest on both emphysema development and SAR is needed in mice in order to comprehensively assess the activities of molecules of interest in these chronic disease processes. 
There are several critical steps in this protocol. First, during the cigarette smoke exposure, it is essential to clean the machine at least once-a-week because tar products deposit in the device which can result in CS exposures that are not sufficient to induce lung disease.&#160; Second, it is essential to inflate lungs well to accurately measure airspace enlargement. If lungs are nicked during their removal from the thorax, this deflates lobes and results in an under-estimation of airspace size. Thus, to protect the lungs, only cut tissue when the tips of the scissor are visible. Third, for accurate analysis of emphysema development stain the alveolar walls of the lung section darkly with Gill&#8217;s stain, and optimize the exposure time for image acquisition to ensure ideal thresholding of the image to generate an accurate binary image for subsequent analysis. Fourth, capture images in a randomized fashion to avoid observer bias, and acquire and analyze at least 20 images per animal to ensure accurate analysis of airspace enlargement which is heterogeneously distributed in CS-exposed C57BL/6 WT mice. Fifth, in the SAR analysis do not make measurements in areas of adventitia that are shared by adjacent bronchi and vessels in broncho-vascular bundles which will lead to an over-estimation of ECM protein deposition around small airways.
There are several limitations of this protocol. First, the relatively long time needed to induce emphysema and SAR in CS-exposed mice (6 months). Second, there are well documented differences in the susceptibility of different strains of mice to develop emphysema in response to CS (10). Third, much less is known about the susceptibility of murine strains other than the C57BL/6 strain to CS-induced SAR. Fourth, the average researcher needs substantial practice in the lung inflation technique in order to consistently obtain optimally inflated lungs for analysis of both emphysema and SAR. 
An advantage of this protocol is that whole&#8211;body cigarette smoke exposure minimizes distress to the mice, and is not labor-intensive to perform (unlike the nose-only exposure method). Compared with other methods, this protocol measures alveolar chord length on randomly-acquired images (as a readout of emphysema), is less time-consuming to complete, less susceptible to observer bias, and more effective at capturing the heterogeneous nature of the pathology in mice as it can analyze the disease in entire lobes and/or both lungs. The protocol also measures both contributors to airflow obstruction (emphysema and SAR) on lung sections from the same mice. 
The protocol enables the researcher to assess the effects of CS on SAR in airways having different sizes. It can be adapted to measure individual ECM protein components deposited around airways in CS-exposed lungs to provide additional information about the nature of the small airway disease in CS-exposed animals. Overall, this protocol can quantify major chronic pulmonary pathologies in the lungs of mice exposed to CS in a relatively automated, accurate, and unbiased manner.

ないモデルが完全ヒト疾患（2）のすべての機能を複製することができないので、COPDを研究するための動物モデルの使用は困難である。ほとんどの研究者は、それらの肺生理学、病理学、遺伝学、および代謝物のマウスとヒトの間の類似性のCOPDをモデル化するために、マウスを使用しています。また、マウスは勉強するのが比較的安価であり、肺気腫、小さな気道リモデリングの両方がCS曝露（5,7-9）の6カ月以内に発症する。 タバコの煙によって誘発されるCOPD：いくつかの方法が、マウスにおいてCOPDを誘発することができる。ほとんどの研究者は、人間のCOPDの主な病因因子であるCS、にマウスを公開。 6ヶ月のCS曝露は、マウスにおいて肺気腫および小さな気道リモデリング（SAR）の開発を引き起こすが、誘導される疾患の重症度を検討マウス株に依存して変化する。 AKR / Jマウスはextremelであるのに対し、例えば、NZWLacZマウスは、CS誘発性肺気腫の発症に耐性である感受性のy（10）。ほとんどの研究者は、多くの遺伝子標的マウスとしてのCS暴露モデルでC57BL / 6系統のマウスを研究この株でご利用いただけます。 CS暴露の6ヵ月後、肺気腫、小気道線維症は、野生型（WT）C57BL / 6マウスで開発し、両方の病変が重症度（5,10）で比較的穏やかです。鼻専用および全身曝露：研究者は、CS暴露の2種類を使用しています。 1）それは、より労働集約的な方法であり：鼻のみの露光技術の主な欠点は、ということです2）マウスを、動物のストレス応答および温熱療法を誘導することができる小室（11）に拘束されなければならない。 （本明細書に記載）の全身暴露の主な欠点は、それらの毛を掃除するとき、動物がニコチンとタール製品を摂取する（同様に吸い込む）ことができることである。全身CSに曝露されたマウスは、より低いヘモグロビンレベルを有し、鼻のみCS（12）に曝露した動物と比較して、体重の損失を減少させた。 肺機能検査（のPFT）：肺コンプライアンスとエラスタンスの措置がこの開発は比較的軽度肺気腫による6ヶ月間空気またはCSに暴露されたC57BL / 6野生型（WT）マウスでは、通常は似ています株は、CS（10）に露出している。気腫性の破壊はより重篤である場合しかし、肺コンプライアンスと圧力 - 体積（PV）の左シフトの増加は、ループを検出することができる流れる。後者は、C57BL / 6 WTマウスよりもより重度の肺気腫型を持つCS曝露C57BL / 6系統の遺伝子標的マウスにおいて、CS（10）の影響を受けやすいマウス株では、例えば、観察することができる（13）、またはCS（14）の影響をより受けやすく、環境の変化にさらさCS曝露マウスで。このプロトコルは、組織における肺の弾性反跳（準静的肺コンプライアンスの増加[Cstに]の削減と削減を測定するための小動物人工呼吸器を使用していますエラスタンス[H]）、PVフローループ、麻酔したマウス（15,16）における気道組織抵抗の変化。 肺気腫の措置：その分布が空間的に不均一であるため、CS-暴露C56BL / 6系統のマウスでの肺気腫の開発の分析は困難である。いくつかの異なる方法は、マウスの空域の拡大を定量化する。使用された最初の方法は、平均線インターセプトさ（L M）（17）であった。しかし、LのM法は（肺のすべてのセクションがランダムにサンプリングされていない限り）疾患の不均一性をキャプチャしない場合があり、その使用は、したがって、分析にオブザーバーバイアスを導入する可能性が遅く、手動のプロセスです。破壊的なインデックス[DI、（18）]は、ヘマトキシリンおよびエオシン染色した肺切片の印刷されたデジタル化された画像の上に置か50均等に分配ポイントを持つ透明シートを使用して空域の拡大を定量化する。 PI法のスコア各ポイントのACを囲む領域この領域内の肺胞管と肺胞壁が破壊される程度にコーディング。 DI方式の主な欠点は、時間がかかり、他の方法（19,20）よりも正確ではないということである。 このプロトコル措置はギルの染料で染色パラフィン包埋肺切片上歯槽弦長と肺胞の領域を意味する。形態計測ソフトウェアは、（組織は白色であり、空域が黒色である）は、バイナリ画像に肺切片の画像を変換し、によって識別領域内の各弦の長さを定量化し、水平線と垂直線（コード）の一様格子とソフトウェアを重畳する空域などのソフトウェア。この方法を使用して、標準化された、比較的自動化方法（21）肺のすべての部分における肺胞の大きさを測定することが可能である。 小気道リモデリング（SAR）：ECMタンパク質沈着の増加（特にinterstitia小気道の周りのLコラーゲンは）CS-暴露動物で発生し、障害物を気流に貢献します。研究者は、肺気腫の開発（22）と同じ頻度でCOPDの動物モデルでSARを勉強しません。 CS曝露マウスにおけるSARを定量化するために、このプロトコルは、パラフィン包埋した肺切片における末梢気道（気道300と899メートルの間の平均直径を有する）の周りに堆積されるECMタンパク質の層の厚さを測定するために画像解析ソフトウェアを使用してマッソン三色染色で染色。

<table><tbody><tr><td>Whole-body smoke exposure device</td><td>Teague Enterprise</td><td>TE-10z</td><td>Chronic Smoke exposures to induce chronic lung disease in mice</td></tr><tr><td>Research Cigarette</td><td>University of Kentucky</td><td>3R4F reference cigarettes</td><td></td></tr><tr><td>Pallflex&amp;reg; Air Monitoring Filters, Emfab Filters TX40HI20WW, 25 mm</td><td>Pall Corporation</td><td>7219</td><td>For measurement of TPMs</td></tr><tr><td>25 mm filter holder</td><td>Pall Corporation</td><td></td><td></td></tr><tr><td>Filter sampler</td><td>Intermatic</td><td>Metal T100</td><td></td></tr><tr><td>Gas meter</td><td>AEM</td><td>Gas meters G1.6; G2.5; G4</td><td></td></tr><tr><td>Tracheal Cannula for mouse 18 gauge</td><td>Labinvention</td><td></td><td>Analysis of pulmonary function</td></tr><tr><td>Mechanical ventilator</td><td>Scireq</td><td>FlexiVent</td><td></td></tr><tr><td>Gill&#039;s hematoxylin solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>GSH316</td><td>For Gill staining, work under a fume hood</td></tr><tr><td>Hematoxylin solution, Harris modified</td><td>Sigma-Aldrich</td><td>HHS16</td><td></td></tr><tr><td>Cytoseal-60</td><td>Thermo Scientific</td><td>8310-16</td><td></td></tr><tr><td>Micro-Slide-Field-Finder</td><td>Andwin Scientific INC</td><td>50-949-582</td><td>For analysis of emphysema</td></tr><tr><td>Scion Image Program</td><td>Scion Corporation</td><td></td><td></td></tr><tr><td>Mason&#039;s trichrome stain</td><td>Sigma-Aldrich</td><td>HT15</td><td>For analysis of small airway fibrosis</td></tr><tr><td>MetaMorp Offline version 7.0</td><td>Molecullar Devices LLC</td><td>31032</td><td></td></tr></tbody></table>

automated measurement of pulmonary emphysema and small airway remodeling in cigarette smoke-exposed mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

このプロトコルは、CSのために、マウスの全身暴露から始まる。 TPMのデバイスと監視の適切な監督やメンテナンスは、一貫性のある喫煙曝露（ 図1）を確保カウントします。研究者は、膨張装置を用いて、肺膨張手法を実践することをことが重要ですこのプロトコルは、CSのために、マウスの全身暴露から始まる。 TPMのデバイスと監視の適切な監督やメンテナンスは、一貫性のある喫煙曝露（ 図1）を確保カウントします。注意深く肺膨張膨張装置を用いる技術（ 図2）とは、空域の拡大の正確な分析のために十分に膨張した肺切片を得るために、膨張後に肺を除去し、研究者の実践することが重要である。図3Bは不十分膨張した肺を示しているのに対し、図3Aはよく膨張した肺を示しています。図3Cは、閾値処理のために調製し、膨張肺切片の画像を示しているTEP（肺胞腔内マクロファージが白く塗られており、血管や気管支がgenerateに黒塗りされている。閾値化ステップは、肺胞腔内の全ての画素が白である、二値画像および肺胞ではない肺の領域内の全ての画素を作成（図3D）黒である。図3E及び3Fは、垂直方向と水平方向の歯槽和音はそれぞれ、マクロが生成することを長さを示している。 肺機能検査（6ヶ月間CSに曝露したC57BL / 6 WTマウスにおいて発症軽度肺気腫と一致する肺の弾性収縮力の適度な損失を反映してループの圧力容積（PV）の左側の適度な（統計的に有意ではない）シフトを示し図4A）。 PVループの有意な左シフトのみCSまたはCS曝露C57BL / 6 WTマウスよりもより重度の肺気腫の表現型を有するCS曝露遺伝子標的マウスにおける効果に非常に敏感であるマウス系統において観察される。 イチジクURE 5は、CS-暴露した動物における末梢気道の周りのECMタンパク質の沈着の増加を示した6ヶ月間の空気（ 図5A）またはCS（ 図5B）に暴露されたC57BL / 6 WTマウスのマッソンの三重染色した肺切片。 図の代表的な画像を示している図5Cは、画像解析ソフトウェアプログラムは、所望の内径を有する気道の周囲のECMタンパク質の沈着を定量化する方法を示す。 図5Dは、CS曝露C57BL / 6 WTマウスにおける300から899ミクロンの直径を有する気道周囲のECMタンパク質沈着の分析を示す。 <img alt="図1" src="/files/ftp_upload/52236/52236fig1highres.jpg" /> 図1.全身シガレット露光システムの漫画。煙曝露装置は煙曝露チャンバーに接続されている。煙は副流収集チャンバから引き出され、煙から引き出されるポンプによるタバコ、および両方の煙試料を混合し、希釈された外気と混合し、希釈室（左）で、その後煙曝露室に流入される。研究者は、露光室（右）におけるそれらのケージにマウスを置く。マウスをケージ内で自由に動くことができる、および煙曝露の持続時間の間、食物および水へのアクセスを持っている。 <img alt="図2" src="/files/ftp_upload/52236/52236fig2highres.jpg" /> ネズミの肺の図2.インフレ。研究者は、滅菌PBSでフラスコを埋めるゴム栓でシールして、それを反転し、それをリングスタンドを用いて動物の心臓部上25cmの距離を確保。静脈寄付セットは、気管カニューレを介して肺にPBSを提供します。カットダウン血清学的ピペットをゴム栓を通して挿入され、これがluにドレインをPBSの体積を置き換えることをフラスコに空気を可能にする重力によるマウスのNGS。 <img alt="図3" src="/files/ftp_upload/52236/52236fig3highres.jpg" /> 図3.肺気腫分析。（A）は Gill&#39;s染色の代表画像は6ヶ月間、空気またはCSに曝露されたマウスからの肺切片を膨らま示し、黒い矢印は、血管や肺胞マクロファージを示している。 （B）は、分析のためには適していない下に膨張した肺の代表的な画像を示す。 （A）は、「前」、および（C）を示している研究者は、二値画像を生成するための準備を代表肺切片の「後」の画像を示している。 （A）及び（C）の黒矢印は血管のいずれかを示す（研究者の黒塗料（C））または肺胞マクロファージ（研究者は、白色塗料（C））。 （D）&lt;<html>研究者がしきい値ステップを実行した後に/ strong&gt;のバイナリイメージを示しています。 （E）及び（F）は、水平および垂直肺胞コードは、それぞれの研究者によって生成される長さを示している。すべての画像の倍率は400ミクロンを表すxの200のスケールバーは、（A）に示されている。 <img alt="図4" src="/files/ftp_upload/52236/52236fig4highres.jpg" /> 図4肺胞弦長及び圧-容積曲線（A）は空気中に露出C57BL / 6 WTマウスにおける肺胞弦長の典型的な分析を示した（n = 13）またはCSた（n = 24）、6ヶ月。アスタリスクは、p &lt;0.001を示している。 （B）は、6ヶ月間空気（N = 13）またはCS（N = 14）に暴露されたC57BL / 6 WTマウスで実行典型的なPVループを示しています。データは平均+ SEMとして表される。 highres.jpg &quot;/&gt; 図5.小気道リモデリング（SAR）査定。 （A）及び（B）は、6ヶ月間空気（A）またはCS（B）に曝露したC57BL / 6 WTマウスからのマッソントリクローム染色した肺切片の代表的な画像を示す。 （C）は、画像解析ソフトウェアは、CS曝露マウスにおけるSARを解析する方法を示している。 （D）は、6のための空気にさらさC57BL / 6 WTマウスにおける300〜899メートルた（n = 11）またはCSた（n = 16）の直径を有する小気道周囲に堆積外マトリックスタンパク質層の厚さの典型的な測定値を示すヶ月。データは、平均値として表される+ SEMアスタリスクは、p &lt;0.05を示す。 スケールバーは、各肺の部分の画像に表示されます。

Watch this Scientific Journal Video about Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice at JoVE.com

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

肺気腫とタバコ煙曝露マウスにおける末梢気道リモデリングの自動測定

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

automated-measurement-pulmonary-emphysema-small-airway-remodeling

Universidad San Sebastian

Research

JoVE Journal

Medicine

13.2K ビュー.  Brigham and Women's Hospital - Harvard Medical School. The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice. 

ビデオ: Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface  

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

エアー液界面におけるマウスの呼吸器上皮細胞と実験たばこの煙への暴露の分離

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

医学

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

アレルゲン誘発性喘息のマウスモデル

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

免疫・感染学

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

気道抵抗測定とマウスにおける気管支肺胞洗浄液のサンプリングのためのリバーシブル、非侵襲的方法

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

生物学

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

マウス肺における圧力 - 容積曲線の測定

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

繰り返しオゾン暴露によるマウスの慢性閉塞性肺疾患モデルの生成

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

強制振動法を用いたマウスにおける呼吸器系の力学の評価

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

ダブル室容積脈波による意識下マウスにおける呼吸機能の評価

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

全身吸入システムを用いたマウスのタバコの煙暴露

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

マウスにおける気腫の評価に対する一定圧力下での肺固定

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.
After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 &#181;m) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 &#181;L of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.
The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

肺上皮間葉転換を促進するためのシリカ曝露マウスモデルでのニコチンの使用

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role ...