Name: generation of a chronic obstructive pulmonary disease model in mice by repeated ozone exposure
Uploaded: 2017-08-25T14:00:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure at JoVE.com

著者はこのプロトコル µCT の評価に関する技術的な支援を療養 Qin 氏 (上海公共衛生臨床センター) に感謝して思います。

注:、純正モデルが生成されて、以前に報告された研究 30 , 31 , 32 で使用します。すべての動物実験は機関動物ケアおよび使用委員会 (IACUC) 上海交通大学によって承認されました。
 1 です。 マウス <ol><li>家病原体フリー、7-9 週齢女性 BALB/c マウスで動物の個別換気ケージ施設制御温度 (20 ° C) と湿度 (40-60%)。12 h 光と施設で 12 h 暗いサイクルを提供します。食品を提供し、広告自由 水します</li>。
</ol> 2。オゾンや大気暴露 <ol><li>シール (例えば 風防) アクリル ボックスに電動発電機とオゾンを生成します。内側に、箱の外側に接続されているエアコン通気管を通じて小さな空気送風機を使用して箱から出して空気を吹きます。オゾン プローブを使用してボックスにオゾン濃度を監視します。ボックスでのオゾンの濃度に達するあたり 2.5 部品までを待つ百万 (ppm) です 。
	注: オゾン プローブ オンまたはオフに自動的にオゾン発生器を切り替えることができます、2.5 ppm のボックスにオゾン レベルを維持できます</li>。
	<li>は、2.5 ppm のオゾンのレベルに達したら、ボックスにマウスを配置します。オゾンにそれらを公開するたびに 3 h のボックスにマウスを保持します 。
	注: ボックスは自動的にオンまたはオフにオゾン発生器を切り替えることにより 3 h 中に 2.5 ppm のオゾン濃度を維持できるし、箱から出してマウスによって生成される CO 2 を吹いています</li>。
	<li>7 週間 (3 日に 1 回) 週 2 つのオゾン露出 (3 時間を持続させるそれぞれの暴露) を与える; 同時にそして同じ期間の空気コントロール マウスを公開します</li>。
</ol> 3。マイクロ コンピューター断層撮影 <ol><li>7 週の終わりには、pelltobarbitalum natricum の腹腔内注入マウスを麻酔 (1%、0.6 - 0.8 ml/100 g) マウスがないことを確認する個々 の状況に応じて投与量を調整つま先ピンチ。 モニターに対応し、安定した呼吸の頻度でマウスを維持手続き中に自主的な動きがないかどうかを確認します</li>。
	<li>マイクロ コンピューター断層撮影 (µCT) の商工会議所に麻酔下のマウスを配置します</li>。
	<li>調整、メーカー標準のプロトコルを使用して µCT &#39; の指示。X 線管を設置 50 kV と 450 μ A の電流 。
	注: 両方、マウスを中心に x 線と検出器回転します</li>。
	<li>µCT 解析を行う 1 つのスライスの 300 ms の露光時間と 0.093 mm スライス厚 0.092 mm 有効画素サイズ 515 予測を取得します</li>。
	<li>は、ソフトウェアを使用して取得した画像で肺を再構築します。それぞれ-750 -550 ハンス台で最小値とグレースケールの最大を設定することでグレースケール画像の明るさを調整します 。
	注: ソフトウェアが自動的に肺実質と低吸収域 (LAA) 34 , 35 のボリュームを計算します</li>。
	<li>LAA ボリューム総肺気量で割ることによって LAA (LAA %) の割合を計算します</li></ol>。
 4 トレッドミル テスト <ol><li>適応マウスの上実行しているトレッドミル マシン注 10 分間 10 m/分の速度でテストを与える: 電気は、常にオフの手順が実施されている場合。</li> <li>管理、。マウスに疲労試験。
	<li>ウォーム アップ 5 分 10 m/分の速度でマウス</li> <ol>
		<li>15 m/分で 10 分間に速度を上げる</li>
		<li>運動強度を高める: 20 m/分、開始 5 m/分まで速度を上げるマウスまで 30 分毎の 36 を実行する続行できません</li>。
	</ol></li> <li>走行距離、距離疲労と疲労時間として時間を実行すると、それぞれの合計を記録します</li></ol>。
 5 肺機能測定 <ol><li>pelltobarbitalum natricum の腹腔内注入マウスの麻酔 (1%、0.6 - 0.8 ml/100 g) マウスを見るのは個々 の状況に応じて投与量を調整。つま先のピンチに応答しないと、マウスは自発呼吸。 モニターを維持し、安定した呼吸の頻度でマウスを維持するを待つ手続き中に自主的な動きがないかどうかを確認します</li>。
	<li>は慎重にマウスを tracheostomize し、コンピューター制御の人工呼吸器に接続されている体プレチスモ グラフでそれらを配置します 。
	注: 換気、気管内チューブに近位に位置する弁を介して制御されます。セットアップは、準静的圧力ボリュームの演習、高速水流量の演習を含む別の半自動操縦を提供します</li>。
	<li>平均通常呼吸パターンまで圧力制御の換気麻酔下マウスに 150 の呼吸/分の呼吸を課すし、各呼吸サイクルの完全な有効期限を取得します</li>。
	<li>プレチスモで生成された負圧を使用して、デバイスと準静的圧力ボリューム操作を実行します</li>。
	<li>は、声帯と fev 社を記録する準静的圧容積ループ内で速い流れボリューム操作を実行します。+30 cm H 2 肺を膨らませる O およびすぐにその後まで残留量-30 cm H 2 o ・ レコードで、最初の 25、50、75 ms 呼気 (fev 社 25 の fev 社は有効期限を設けるに高い負圧に接続、Fev 社 50、と fev 社 75、それぞれ)。最適な演習を拒否します。ひとつひとつのマウスと各テストでは、すべての数値パラメーターの信頼できる平均を取得する 3 つの許容される作戦の最低です</li>。
</ol> 6。BALF コレクション <ol><li>pelltobarbitalum natricum ターミナル麻酔後 ((1%、1.8 2.4 ml/100 g)、マウスはないつま先ピンチに対応し、息を失うことを参照してくださいに個々 の状況に応じて投与量を調整)、洗浄、2 ml の PBS の 1 mm の気管内チューブと、取得 BALF 10 を介してマウスします</li>。
	<li>取得した洗浄因数をプール、それら 4 ° C で 250 x g で 10 分間遠心</li>
	<li>即座の使用のための上澄みを集め、-80 ° C または液体窒素で残りの部分を格納します</li>。
	<li>診断を使用してセルの合計数をカウントします</li>。
	<li>PBS で、スピン (1,400 x g、6 分) 250 μ L のスライド スピンの遠心分離機を使用してスライド上に細胞を再懸濁細胞ペレットを再懸濁します</li>。
	<li>製造業者に従ってスライド細胞適用ライト染色 &#39; s プロトコル</li>。
	<li>マウスあたり 200 セルをカウント; 400 倍の倍率での標準的な形態によるとマクロファージや好中球、セルを識別し、その番号をカウントします</li>。
</ol> 7。心臓採血 <ol><li>心臓穿刺による採血、1.5 mL チューブにそれを読み込む、氷上で 30 分間それを維持</li>
	<li>G × 2,000 と 4 で 5 分間の血液サンプルを遠心分離機 ° C</li>
	<li>新しいチューブに上清 (血清) を転送し、-80 ° C または液体窒素で保存します</li>。
	<li>Il-1 β の血清を準備 IL-10 ・ TNF α 検出テストのそれぞれの ELISA キットを使用しています</li>。
</ol> 8。肺木股 <ol><li>マウスから気管、肺を解剖。
	<ol><li>犠牲後すぐに手術のボードの上にそれぞれの euthanized マウスを配置します</li>。
		<li>広頸筋を離れて分析と視覚化し、気管軟骨輪をアクセスする前脛骨気管筋</li>。
		<li>、胸部を開く空洞。解剖肺と、気管が肺から心臓を区切らないでください</li>。
	</ol>
</li> <li>PE90 ポリエチレン管を通って 4% パラホルムアルデヒドの入った注射に気管内カテーテルを接続します 。
	注意: パラホルムアルデヒドは有毒です。手袋と安全メガネを着用、ヒューム フード内部ソリューションを使用します</li>。
	<li>は、気管内カテーテルを 4% パラホルムアルデヒド (10 滴、〜 200 μ L) を使用して完全に肺を膨らませます。インフレの完了後中心部を削除します</li>。
	<li>少なくとも 4 時間のための 4% のパラホルムアルデヒドの 10 mL を含む 15 mL チューブに肺を維持</li>
	<li>はパラフィンに肺を埋め込みます。回転式ミクロトームで、セクショニング パラフィン ブロックで 5 μ m のセクションを取得します。断面、肺の気管支領域内の最大の表面積を公開します</li>。
	<li>形態計測学的解析、実行ヘマトキシリンとエオシン (H &#38; E) セクションに染色します</li>。
	<li>明るいフィールド正立顕微鏡 (対物レンズ 20 X; 露光時間、1.667 ms) とセクションのイメージします</li>。
	<li>がある 2 つの調べ治療プロトコルに目がくらんで組織切片を別々 にカウントします。間肺胞中隔の壁までの距離を測定するためのパラメーターとして平均線形インターセプト (L m) を使用します。L m 次の手順を使用してを決定する: <ol><li>セクションのイメージを Photoshop で開き、5 550 μ m の長い行のイメージにレティクル グリッドを描画します</li>。
		<li>グリッド線上に肺胞の数をカウントします</li>。
		<li>は、グリッドの行の長さを肺胞の数で割ることによって L m を計算します。定量化, 画像のマウスごとの 5 つのセクションを実行します。各セクション (フィールド 1 つのイメージ) の 10 の画像を取得し、ランダムに評価します。フィールド選択中に前方または別の方向に 1 つのフィールドを移動することによって航空・船舶分野を避ける 。
		注: データは、平均 ± S.E.M. として記載して非対応の t 検定は空気にさらされたマウスとマウスのオゾン暴露間の比較を行った。各グループの 3 つの動物は、有意の差の計算に使用されました。P-値 &#60; 0.05 が重要視され</li>。
	</ol>
	</li>
</ol>

Z.W.S. と大戦は、現在の従業員と細胞生物医学グループのストック オプションの保有者 (NASDAQ: CBMG)。他の作家は、彼らが競争の興味あることを宣言します。

この研究では、COPD の新しいモデルを生成するための信頼性の高い方法を提案する.この OE モデル (すなわち、 LPS または PPE モデル) の他のモデルと比較して、COPD 患者の病理学的プロセスを繰り返します。タバコの煙は、人間の患者40で COPD を引き起こす主な有害物質は、最も人気のある COPD モデル41,42のまま CS モデルです?...

それは、COPD、肺気腫と慢性気管支炎を含むかもしれない 2020年1,2の世界の死の 3 番目の主要な原因をこと推定されています。人口の 40 歳以上の COPD の潜在的な発生率は、男性で 12.7%、次の 40 年の3で女性の 8.3% と推定されます。COPD 患者4の進行悪化を逆に現在利用できる薬はありません。COPD の信頼性の高い動物モデルだけでなく病気の病理学的プロセスの模倣の需要、また短い世代期間が必要。LPS または PPE 誘発モデルを含む、現在の COPD モデルは、肺気腫のような症状5,6を引き起こすことができます。単回投与または LPS または PPE の一週間にわたる課題著明な BALF の血清、プロ炎症性メディエーター (例えばTNF-α や il-1 β) を増加、気管支肺胞洗浄液 (BALF) でマウスやラットの結果を生成する肺実質の破壊拡大の空気のスペースと制限気流5,6,7,8,9,10。ただし、組合や PPE 人間 COPD の原因ではない、従って11病理学的プロセスを模倣しません。CS 誘起モデルは、永続的な気流制限、肺実質の破壊を生産し、機能的運動能力を削減します。しかし、伝統的な CS プロトコルには、COPD モデル12,13,14,15を生成するため、少なくとも 3 ヵ月が必要です。したがって、2 つの要件を満たしている新しいより効率的な動物モデルを生成する重要です。
最近では、タバコを吸う、に加えて大気汚染や職業暴露は COPD16,17,18のより一般的な原因になっています。主要な汚染物質の一つとして、オゾン (ただし大気汚染の主要なコンポーネントではなく)、することができます直接気道反応し、子供や若い大人19,20,21 の肺組織の損傷 ,22,23,24,25。組合、PPE は、CS などその他の刺激と同様に、オゾン、深刻な肺の酸化ストレスや DNA の損傷の生化学的経路に関与しているし、開始と COPD26,27のプロモーションにリンクしています。別の要因は、オゾン、オゾンが肺機能18,28,29を破壊できることを示すにさらされた後いくつかの COPD 患者の症状が悪化することです。したがって、我々 は繰り返し 7 週間のオゾンの高濃度にマウスを公開することによって新しい COPD モデルを生成これは気流の欠陥や肺実質損傷前調査30,31,32のものと同様で起因しました。本研究では雌マウスに OE プロトコルを拡張し、私たち以前研究30,31,32の雄マウスにみられる肺気腫を再現します。COPD 死亡率は男性で減少したが、多くの国の33の女性で増加、ためメカニズムの研究と女性の COPD 患者の治療法を開発する女性の COPD モデルが必要です。すべての性別に OE モデルの適用性は、COPD モデルとしての使用にさらにサポートを貸します。

<table><tbody><tr><td>BALB/c mice</td><td>Slac Laboratory Animal,Shanghai, China</td><td>N/A</td><td>7-to-9-week-old female BALB/c mice were used in this study.</td></tr><tr><td>Individual ventilated cages</td><td>Suhang, Shanghai, China</td><td>Model Number: MU64S7</td><td>The cages were used for housing mice in the animal facility.</td></tr><tr><td>Sealing perspex-box</td><td>Suhang, Shanghai, China</td><td>N/A</td><td>The box was used&amp;nbsp; to contain the ozone generator. Mice were exposed to ozone within the box.</td></tr><tr><td>Electric generator</td><td>Sander Ozoniser, Uetze-Eltze, Germany</td><td>Model 500&amp;nbsp;</td><td>The device was used for generating ozone.</td></tr><tr><td>Ozone probe</td><td>ATi Technologies, Ashton-U-Lyne, Greater Manchester, UK</td><td>Ozone 300</td><td>The device was used for monitoring and controlling the generation of ozone.</td></tr><tr><td>Pelltobarbitalum natricum</td><td>Sigma, St. Louis, MO, USA</td><td>P3761</td><td>Mice were anesthetized by intraperitoneal injection of pelltobarbitalum natricum.</td></tr><tr><td>Micro-Computed Tomography</td><td>GE Healthcare, London, ON, Canada</td><td>RS0800639-0075</td><td>This device was used for acquiring images of the lung.</td></tr><tr><td>Micro-view 2.01 ABA software</td><td>GE Healthcare, London, ON, Canada</td><td>Micro-view 2.01&amp;nbsp;</td><td>This device was used for reconstruct the lung and analyze volume, LAA of the lung.</td></tr><tr><td>Treadmill machine&amp;nbsp;</td><td>Duanshi, Hangzhou, Zhejiang, China</td><td>DSPT-208</td><td>This machine was usd for fatigue test.</td></tr><tr><td>Body plethysmograph</td><td>eSpira&amp;trade; Forced Manoeuvres System, EMMS, Edinburgh, UK</td><td>Forced Manoeuvres System</td><td>This device was used to test spirometry pulmonary function.</td></tr><tr><td>Ventilator</td><td>eSpira&amp;trade; Forced Manoeuvres System, EMMS, Edinburgh, UK</td><td>Forced Manoeuvres System</td><td>This device was used to test spirometry pulmonary function.</td></tr><tr><td>Slide spinner centrifuge</td><td>Denville Scientific, Holliston, MA, USA</td><td>C1183&amp;nbsp;</td><td>It was used to spin BALF cells onto slides.</td></tr><tr><td>Wright Staining</td><td>Hanhong, Shanghai, China</td><td>RE04000054</td><td>&amp;nbsp;It was used to staining macrophages, neutrophils in the suspended BALF.</td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific, Horsham, PA, USA</td><td>4000</td><td>It was used to count cells.</td></tr><tr><td>IL-1&amp;beta;</td><td>Abcam, Cambridge, MA, USA</td><td>ab100704</td><td>They were used to test the respective factors in serum.</td></tr><tr><td>IL-10</td><td>Abcam, Cambridge, MA, USA</td><td>ab46103</td><td>They were used to test the respective factors in serum.</td></tr><tr><td>TNF-&amp;alpha;</td><td>Abcam, Cambridge, MA, USA</td><td>ab100747</td><td>They were used to test the respective factors in serum.</td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma, St. Louis, MO, USA</td><td>P6148</td><td>The lung was inflated by 4% paraformaldehyde.</td></tr><tr><td>Paraffin</td><td>Hualing, Shanghai, China</td><td>56#</td><td>It was used to embed the lung.</td></tr><tr><td>Rotary Microtome</td><td>Leica, Wetzlar,&amp;nbsp; Hesse, Germany</td><td>RM2255</td><td>It was used for sectioning the lung.</td></tr><tr><td>Hgaematoxylin and Eosin (H&amp;amp;E) staining solution</td><td>Solarbio, Beijing, China</td><td>G1120</td><td>H&amp;amp;E staining was done for morphometric analysis.</td></tr><tr><td>Upright bright field microscope</td><td>Olympus, Center Valley, PA, USA</td><td>CX41</td><td>It was used to image the H&amp;amp;E staining slides.</td></tr><tr><td>Adobe Photoshop 12</td><td>Adobe, San Jose, CA, USA</td><td>Adobe Photoshop 12</td><td>It was used to count the number of alveoli on the H&amp;amp;E stained images.</td></tr><tr><td>GraphPad prism 5</td><td>Graphpad Software Inc., San Diego, CA</td><td>GraphPad prism 5</td><td>It was used for data analysis and production of figures.</td></tr></tbody></table>

generation of a chronic obstructive pulmonary disease model in mice by repeated ozone exposure

慢性閉塞性肺疾患 (COPD) は、永続的な気流制限や肺実質の破壊によって特徴付けられます。それは人口高齢化における発生率が非常に高いです。COPD フォーカスを中心に症状変更薬; 現在の従来の治療法したがって、新しい治療法の開発が急務します。COPD の修飾の動物モデルは基になるメカニズムを特徴付けるために助けることができるし、新しい薬物スクリーニングに使用することができます。リポ多糖 (LPS) など、現在の COPD モデルやブタ膵臓エラスターゼ (PPE)-誘導肺気腫モデル肺と気道における COPD のような病変を生成が、そうでなければ人間 COPD の病因を似ていません。タバコの煙 (CS)-誘導モデルのまま 1 つの最も人気のあるのでそれはだけでなく呼吸器系における COPD のような病変をシミュレートしますが、それは人間の COPD を引き起こす主な有害物質の一つに基づいています。ただし、CS 誘発モデルの時間のかかり、労働集約的な側面は、劇的に新しい創薬スクリーニングへの応用を制限します。この研究では、正常に高濃度のオゾンにマウスを公開することによって新しい COPD モデルを生成しました。このモデルは次を示した: 1) 強制呼気量 25、50、75/強制肺活量の減少 (fev 社25/FVC、fev 社50/FVC と fev 社75/FVC)、肺機能の悪化を示す2) 拡大肺肺胞、肺実質の破壊;3) 削減疲労時間と距離。・ 4) 炎症を増加します。一緒に取られて、これらのデータは、オゾン暴露 (OE) のモデルがオゾン露出オーバーは COPD の病因の要因の一つは、人間に同じような信頼性の高い動物モデルであることを示します。さらに、6-8 週間、OE モデル COPD 研究の良い選択であるかもしれないことを示すタバコの煙モデルを誘発する 3-12 ヶ月を必要とするのに対し、純正モデルを作成する私たちの前の仕事に基づくだけかかった。

各グループの 3 D µCT 画像の例としては、図 1に表示されます。オゾン暴露マウスが大きく総肺気量 (図 1と b) と LAA % (図 1c) 空気露出コントロール マウスよりも。肺気量と LAA % に残ったオゾン露出31,32の 6 週後上昇。LAA ...

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

本研究では、オゾンの高濃度にマウスを繰り返しさらすことによって新しい慢性閉塞性肺疾患 (COPD) の動物モデルが正しく生成について説明します。

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 ...

generation-chronic-obstructive-pulmonary-disease-model-mice-repeated

Research

JoVE Journal

Medicine

10.9K ビュー.  Cellular Biomedicine Group, Shanghai. 本研究では、オゾンの高濃度にマウスを繰り返しさらすことによって新しい慢性閉塞性肺疾患 (COPD) の動物モデルが正しく生成について説明します。

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

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

長期的な慢性<em&gt;緑膿菌</emマウスにおける&gt;気道感染

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

免疫・感染学

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.

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 ...

医学

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

肺のマイクロ Rna 発情周期にわたってプロファイリングのオゾン暴露マウスの

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

オゾン暴露後の肺胞マクロファージエフェロサイトーシスの生体内評価

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

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 ...

生物学

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 ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

オゾンとLPS誘発マウス急性肺損傷時の肺細胞適応の可視化

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and treatment of silicosis in humans have all been extensively studied using the mouse silicosis model. By repeatedly making mice inhale CSD into their lungs, the mice can mimic the clinical symptoms of human silicosis. This methodology is practical and efficient in terms of time and output and does not cause mechanical injury to the upper respiratory tract due to surgery. Furthermore, this model can successfully mimic acute/chronic transformation process of silicosis. The main procedures were as follows. The sterilized 1-5 &#181;m CSD powder was fully ground, suspended in saline, and dispersed in an ultrasonic water bath for 30 min. Mice under isoflurane-induced anesthesia switched from shallow rapid breathing to deep, slow aspiration for approximately 2 s. The mouse was placed in the palm of a hand, and the thumb tip gently touched the lip edge of the mouse's jaw to straighten the airway. After each exhalation, the mice breathed in the silica suspension drop by drop through one nostril, completing the process within 4-8 s. After the mice's breathing had stabilized, their chest was stroked and caressed to prevent the inhaled CSD from being coughed up. The mice were then returned to the cage. In conclusion, this model can quantify CSD along the typical physiological passage of tiny particles into the lung, from the upper respiratory tract to the terminal bronchioles and alveoli. It can also replicate the recurrent exposure of employees due to work. The model can be performed by one person and does not need expensive equipment. It conveniently and effectively simulates the disease features of human silicosis with high repeatability.

This protocol describes a method for establishing a mouse model of silicosis through repeated exposure to silica suspensions via a nasal drip. This model can efficiently, conveniently, and flexibly mimic the pathological process of human silicosis with high repeatability and economy.

結晶性シリカダストの反復吸入による珪肺症マウスモデル

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and ...

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse models continue to be an important tool for developing new anti-fibrotic strategies. In this study, intrapulmonary administration of bleomycin (BLM) is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimics clinical disease characteristics. C57BL/6 mice received BLM (7 U/mL, 30 min/day) by nasal nebulization for 3 consecutive days and were sacrificed on day 9, 16, or 23 to observe inflammatory and fibrotic changes in lung tissue. Nasal aerosolized BLM directly targeted the lungs, resulting in widespread and uniform lung inflammation and fibrosis. Thus, we successfully generated an experimental mouse model of typical human pulmonary fibrosis. This method could easily be used to study the effects of the administration of various nasal aerosols on lung pathophysiology and validate new anti-inflammatory and anti-fibrotic treatments.

<meta charset="utf8"></head><body>鼻ブレオマイシン噴霧により誘発される肺線維症のマウスモデル

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

鼻ブレオマイシン噴霧により誘発される肺線維症のマウスモデル

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse ...