Name: a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation
Uploaded: 2017-07-26T15:00:00.000+00:00
Duration: 8 min 46 s
Description: Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

著者は北京生物科学研究所に支援を求めています。この研究は、北京市自然科学財団（No Z17110200040000）の支援を受けました。

このプロトコールで使用される全ての手順は、北京生物科学研究所の実験動物のケアおよび使用のためのガイドラインの推奨に従って行われた。実験が実施されるまで、8週齢のCD-1雄マウスを特定病原体不使用（SPF）施設に収容した。手術は、完全に麻酔したマウスを使用して行った（ すなわち 、つま先のつまみの反応なし）。手術後、マウスは、十分な食物および淡水を有する温かく湿った室内に置かれた。マウスを、腹腔内注射により送達された過剰の麻酔薬を用いて犠牲にした。 1.マウスPNX手術<ol><li>腹腔内（IP）注射によりナトリウムフェノバルビタール（120mg / kg体重）およびブプレノルフィン（0.1mg / kg体重）でマウスを完全に麻酔する。マウスがつま先へのピンチに反応しなくなったときに手術を行います。 </li><li>ケミカルデポを用いてマウスの左胸部の毛を除去する（約3×3cm 2面積）の治療を必要とする。 </li><li>各マウスを腹側を操作者に向けて挿管台に固定する（ 図1A ）。 </li><li>マウスの舌を引き出し、カテーテル18 （ 図1A ）を案内するノッチを含む小さな動物の喉頭鏡で声帯を照らす。 </li><li>呼吸中に声帯の動きを観察して声帯を区別します。静かに20Gの静脈内挿管カニューレを前角19で気管に挿入する。 </li><li>マウスを右横臥位に置き、カニューレを機械的人工呼吸器（ 例えば、圧力制御; 表の表を参照）に接続する。マウスの胸部の呼吸運動を観察して、カニューレの気管への挿入を確認します（ 図1B ）。 </li><li>吸息圧力を設定する換気装置を12 cmH 2 Oに設定し、呼吸速度を毎分120回の呼吸に設定します （ 図1B ）。 </li><li>ベタジンと70％エタノールで外科領域の皮膚を汚染除去する。 </li><li> Noyes Spring Scissors（刃先14 mm、先端直径0.275 mm）（ 図2B 、C ）を用いて、皮膚と筋肉を切断して、5 番目の肋間腔の空間に2〜3 cmの後外側開胸切開を行う。開胸手術に使用される手術器具は、使用前に滅菌される。 </li><li> 第 5肋間腔で1.5cmの切開を行い、左肺を露出させる（ 図2D 、E ）。操作中は、出血を止めるために高温焼灼器を​​使用してください。 </li><li>左肺葉の3分の1を鈍い先端鉗子で胸から持ち上げ（ 図2F ）、次に綿棒を使用して左全体を引き出す肺（ 図2G ）。 </li><li>左肺葉の肺動脈および気管支を特定する（ 図2G ）。 </li><li> hilumの気管支と血管を絹の外科用縫合糸でしっかりと結紮し、結紮から3〜4mmで左肺葉を切り取る（ 図2H 、I ）。 注：気胸（ すなわち 、胸部の空洞内の空気またはガス）を引き起こす可能性のある左丘の縫合結び目を切断しないように注意してください。 </li><li> 1つの縫合糸で胸壁を閉め、筋層と皮膚層を5〜6本の中断縫合糸を用いて縫い合わせる。各縫合糸の間に3〜4 mmの隙間を残す（ 図2M 、2N ）。 注：外科用縫合針を心臓から離してください。不慮の心臓穿刺は即座に死に至る。 </li><li>ポビドンヨードで手術部位を消毒する。 </li><li>アフト外科手術で38℃のサーマルパッドにマウスを置き、自発呼吸の動きが始まるまでマウスを人工呼吸器に接続します（ 図2O ）。 </li></ol> 2.プロテーゼインプラント<ol><li> PNX処置のステップ1.1〜1.13を実行します（つまり、マウスの左肺葉を取り除いた時点まで）。 </li><li>鈍い鉗子（ 図2J ）を使用して、シリコンプロテーゼの中心（顧客が作成した、長さ12mm、厚さ3mm、幅7mm、0.2g、楕円形）を締め付けます。シリコーンプロテーゼを挿入する前に滅菌する。 </li><li>胸腔を露出させるために片手で鉗子でリブを保持し、もう一方の手で左の空の胸腔にプロテーゼを挿入します。 注：挿入角度は、プロテーゼの前面と胸郭面との間で約45度です（ 図2K 、L）。プロテーゼを挿入するときは非常に優しい。過度の力は胸膜破裂をもたらす。 </li><li>鈍い鉗子でプロテーゼの向きを調整して、プロテーゼが左の空の胸腔を占めるようにします。 </li><li>マウスPNX手順のステップ1.14-1.16を実行します。 </li></ol> 3.ILVの測定<ol><li>使い捨ての血清学的ピペット（10 mL）から取り出したプランジャー、針アダプター、流量制御バルブ、および18 G針を備えた長さ40 cmのフレキシブルチューブからなるカスタムデバイス（「膨張チューブ」）を準備します。組み立て後、テープでボードにピペットを固定します （ 図3A ）。ピペットの上部と実験台との間の距離は、少なくとも30cmでなければならない。 </li><li> 55℃の水浴中で予め加熱した1×リン酸緩衝生理食塩水（PBS）500mLにPFA 20gを溶解し、手動で振とうして新鮮な4％パラホルムアルデヒド（PFA）固定液を調製する溶液が透明になるまで10分おきに静置する。室温に冷却した後、溶液を0.45μmフィルターで濾過する。 注意：PFAを取り扱うときは、適切な個人用保護具（PPE）を着用してください。 </li><li>マウスを麻酔薬の過剰投与（0.8％のフェノバルビタールナトリウム、1,000U / mLヘパリン）で犠牲にする。 </li><li>ポリスチレン解剖プレートに各マウスを固定し、70％アルコールを噴霧する。 </li><li>注意深くマウスの胸を開き、はさみを使用して胸骨を切り開いて肺葉を完全に露出させます。 </li><li>はさみを使用して過剰な組織を除去し、気管を露出させる。食道から気管を離してください。 </li><li>腹部大動脈を切断し、心臓の右心室に25ゲージの針を挿入する。この挿入の前に針を20 mLシリンジに接続してください。肺が白くなるまで、ゆっくりと1x PBSを心臓に押し込み、血球を除去します。典型的には、5~10mLのPBSが肺血管を浄化するために必要とされる。 </li><li>あなたを埋める4％の新鮮なPFAを含むカスタムビルドのインフレーションチューブを取り出し、インフレーションチューブからすべての泡を除去します。 </li><li>インフレーションチューブの18ゲージの針を気管に挿入し、液体漏れを防ぐために気管クリップで気管をクリップする。 </li><li> 25cmで一定経肺圧力/ H 2 O 2、20℃で4％PFAで肺を膨らませます。完全に拡張した肺を達成するために、肺を室温で2時間インキュベートする。この「予備固定」工程は、肺の形態を維持するために重要である。 <ol><li>膨張チューブを監視することにより、最初の4％PFA量の値を記録し、最終量を記録する。内部肺容積は、初期4％PFA容積から最終4％PFA容積を差し引いた容積に等しい。 </li></ol></li><li>気管をLigateし、はさみを使用して、周囲の結合組織から肺を静かに（肺をそのままに）解剖する。肺の損傷を避けるために、非常に穏やかにしてください。 </li><li>インコ4％PFAを満たした50 mLコニカルチューブで、4℃で12時間、シェーカー（50 rpm）で穏やかに振とうしながら肺を栓をする。組織の処理と染色に進む（セクション4を参照）。 </li></ol>組織埋め込み、切片化、およびヘマトキシリン＆エオシン（H＆E）染色<ol><li>固定後、Noyes Spring Scissorsを使用して、肺から心臓や過剰な結合組織を整えます。肺葉を気管に接続する気管支を切断することによって、個々の肺葉を静かに分離する。 </li><li>オービタルシェーカー（50 rpm）で50 mL 1x PBS（30分/洗浄）で3〜4回肺葉を広範囲に洗浄します。 </li><li>最後の洗浄後、組織が50mLコニカルチューブ（約12時間）の底に沈むまで4℃で30％ショ糖溶液（1×PBS）に浸すことによって肺葉を凍結保護する。 </li><li>組織を包埋して凍結切除する前に、鉗子で肺葉試料を管から取り出し、アクセスを保持する約30分間、最適切断温度（OCT）化合物を含有するペトリ皿に試料を完全に浸漬する。 </li><li>クライオホルムを使用して液体窒素中でOCTに埋め込まれたアクセサリーローブサンプルを凍結させる。ローブの最大表面積をモールドの底面に平行にします。 </li><li>組織学的分析のために凍結切片作製中に、各試料について合計3つの厚さ10μmの切片を調製する。最初の1mmの組織を捨て、10μmの厚さの部分を1枚集め、0.5mmの組織を捨て、別の部分を集め、0.5mmの組織を捨て、第3の（最終）部分を集める。 </li><li> H＆E染色を行う前に、セクションを1時間風乾します。 </li><li> H＆E染色を行う <ol><li>水道水の3〜4回の切替えで切片を洗浄し、新鮮なヘマトキシリン中の切片を2分間染色する。;実行中の水道水の下でセクションをすすぎます。切片を1％HCl-70％エタノール溶液に2回浸して過剰のヘマトキシリンを除去する。 </li><li>新鮮なエオシンの切片を3分間染色する。 95％エタノール中で2回連続して30秒洗浄し、100％エタノールで2回30秒洗浄することにより切片を脱水する;キシレン中の切片を30秒間透明にし、清澄化工程を新鮮なキシレン中で1回繰り返す;カバーガラスを使用してスライドをマウント媒体でマウントします。 </li></ol></li></ol> MLIの定量化<ol><li>明視野顕微鏡を用いて、H＆Eで染色されたアクセサリーローブ切片（20倍の倍率）のデジタル画像を取得する。 </li><li> MLIを定量化するには、3つのセクションからなる適切な領域（動脈および静脈、大気道および肺胞管なし）から無作為に合計15個の非重複ビュー（1,000μmx1,000μm）を選択します。 </li><li> 10個の均等に配分された垂直線と定義されたlenの均等に分布した水平線10個でグリッドを配置する選択された領域にルーラーツールを使用してgth（1,000μm）を表示します。したがって、各ラインは100μm離れて配置されている（ 図4B ）。 </li><li> 1つの切片の値を、2つの隣接する肺胞上皮の間の線形長さとして定義する。各1,000μmの長さのラインに沿ったすべての切片の値を測定する。 </li><li>各グリッドについて、10本の水平な1,000μmの長さのラインと10本の垂直な1,000μmの長さのラインの間のすべての切片の値を定量化する。 注：MLIは、アクセサリーローブのそれぞれについて用意された3つのセクションの中から分析された合計15個のグリッドからの切片長の平均値である。 </li></ol> ISAの計算<ol><li> 式1を使ってISAを計算します（ はじめにを参照）。 ILVの測定についてはセクション3を参照し、MLIの定量化についてはセクション5を参照してください。 </li></ol>

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C., 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=Detection+of+murine+post-pneumonectomy+lung+regeneration+by+18FDG+PET+imaging.">Detection of murine post-pneumonectomy lung regeneration by 18FDG PET imaging.</a> EJNMMI Res. 2 (1), (2012).</li><li>Mu&#241;oz-Barrutia, A., Ceresa, M., Artaechevarria, X., Montuenga, L. M., Ortiz-De-Solorzano, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+lung+damage+in+an+elastase-induced+mouse+model+of+emphysema.">Quantification of lung damage in an elastase-induced mouse model of emphysema.</a> Int J Biomed Imaging. 2012, (2012).</li></ol>

著者は何も開示することはない。

このプロトコールでは、マウス左肺PNXおよび人工器官移植後の肺パラメータの測定についての詳細な説明を提供する。 ISAは現在、多くの肺疾患および傷害誘発性肺胞再生における呼吸機能の評価のための重要なメトリックであると考えられている。しかし、肺研究の共同体はISAの有用性について有用な指標として合意されているが、ISAを計算するために使用されるILVとMLIの測定の標準化につ...

肺の基本的機能は、血管と大気との間の酸素と二酸化炭素の交換である。気管支肺胞形成異常（BPD）、慢性閉塞性肺疾患（COPD）、急性呼吸器感染症などの肺疾患は、ISA 2の減少をもたらす。肺疾患を研究研究者はMLI、ILV、ガス交換ユニットの数、ISA、および肺組織のコンプライアンス2、3などの肺の形態学的変化を評価するためにいくつかの定量的な方法を開発しました。 Weibel らによる先駆的研究4およびDuguid ら 5一緒にISAは、ヒト肺における肺のガス交換能力の直接の尺度として使用することができ、肺気腫の重症度を決定するための基準として使用することができることを確立しました。過去5年間に発表された多くの研究では、肺の形態学的パラメーター（ 例えば、 </e開発6中や傷害PNX 1、7から回復中のマウスの肺の形態学的および機能的変化を評価するために、M&gt; ISAとMLI）。 ISAは、 式（1）8,9用いて計算されます。 <img alt="式" src="/files/ftp_upload/56114/56114eq1.jpg" />ここで、ILVは、内部肺容積であり、MLIは、肺周辺空隙サイズ10を表す中間パラメータである。 PNX、一つ以上の肺葉の外科的切除は、広く人間11、マウス1、犬12、ラット13、およびウサギ14、15を含め、多くの種で、肺胞の再生を誘導することが報告されています。スタッドPNX後14日目にマウスの肺のyは、既存の肺胞の拡大と肺胞の新形成の両方が、残りの肺組織におけるISA、ILV、および肺胞の数の回復に寄与することを示した1 。我々と他の人は、スポンジ、ワックス、カスタム形状のプロテーゼなどの材料を空の胸腔にPNX（ すなわち人工器官の植え込み）後に挿入すると、肺胞の再生が損なわれることを示しています。今ではしっかりと肺胞の再生1、16、17を開始するための最も重要な要因の一つとして、その機械的な力の機能を確立しています。そのような研究は、肺胞再生を定量的に評価する基準として、PNX処置および人工器官埋め込み肺からのISA値を使用する有効性を強調している。 観察者バイアスは、測定されたvaに有意に影響を及ぼすことが知られている肺の形態学的パラメーター（ 例えば 、MILおよびILV）についてのルーツ。 ISAの計算に使用される2つのパラメータであるILVとMLIの両方を決定する際に、標準化されたプロトコルを使用してこのバイアスを回避することができます。ここでは、これらの肺パラメータを測定するための非常に詳細な、標準化されたプロトコルを提供します。重要なことに、ISAを正確に定量する能力は、損傷誘発性の肺胞再生モデルにおける肺機能の研究の信頼性および再現性を改善することを約束し、複数の肺疾患における機械的発見を促進するはずである。

<table><tbody><tr><td>Low cost cautery kit</td><td>Fine Science Tools</td><td>18010-00</td><td></td></tr><tr><td>Noyes scissors</td><td>Fine Science Tools</td><td>15012-12</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Castroviejo Micro Needle Holders</td><td>Fine Science Tools</td><td>12060-01</td><td></td></tr><tr><td>Vessel clips</td><td>Fine Science Tools</td><td>18374-44</td><td></td></tr><tr><td>I. V. Cannula-20 gauge</td><td>Jinhuan Medical Product Co., LTD.</td><td>29P0601</td><td></td></tr><tr><td>Surgical suture</td><td>Jinhuan Medical Product Co., LTD.</td><td>F602</td><td></td></tr><tr><td>Mouse intubation platform</td><td>Penn-Century, Inc</td><td>Model MIP</td><td></td></tr><tr><td>Small Animal Laryngoscope</td><td>Penn-Century, Inc</td><td>Model LS-2-M</td><td></td></tr><tr><td>TOPO Small Animal Ventilator</td><td>Kent Scientific</td><td>RSP1006-05L</td><td></td></tr><tr><td>Thermal pad</td><td>Stuart equipment</td><td>SBH130D</td><td></td></tr><tr><td>Pentobarbital sodium salt</td><td>Sigma</td><td>P3761</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>Hematoxylin Solution</td><td>Sigma</td><td>GHS132</td><td></td></tr><tr><td>Eosin Y solution, alcoholic</td><td>Sigma</td><td>HT110116</td><td></td></tr><tr><td>10 mL Pipette</td><td>Thermo Scientific</td><td>170356</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>O.C.T Compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>cryosection machine</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>Disposable Base Molds</td><td>Fisher HealthCare</td><td>22-363-553</td><td></td></tr><tr><td>18 gauge needle</td><td>Becton Dickinson</td><td>305199</td><td></td></tr><tr><td>Povidone iodine</td><td>Fisher Scientific</td><td>19-027132</td><td></td></tr><tr><td>70% ethanol</td><td>Fisher Scientific</td><td>BP82011</td><td></td></tr><tr><td>Infusion sets for single use</td><td>Weigao</td><td>SFDA 2012 3661704</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Tapes</td><td>3M Scotch</td><td>8915</td><td></td></tr><tr><td>Cotton pad</td><td>Vinda</td><td>Dr.P</td><td></td></tr><tr><td>Silicone prosthesis</td><td>Custom made</td><td></td><td></td></tr><tr><td>Brightfield microscope</td><td>Olympus</td><td>VS120</td><td></td></tr><tr><td>Ruler tool</td><td>Adobe Photoshop</td><td></td><td></td></tr></tbody></table>

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.

<html>ここでは、PNX治療群と人工器官埋め込み（義肢植込み）群の実験を行った。これらのグループ分けは、研究グループ14の以前に発表された研究で使用されたグループ分けと同じです。 マウスPNXおよびプロテーゼ移植手順を図2に示す。 8週齢のCD-1雄マウスを手術および定量のた?...

Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

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

内臓の肺表面積（ISA）は、肺疾患および傷害誘発性肺胞再生における肺の形態学および生理学を評価するための決定的な基準である。ここでは、肺の肺切除術とプロテーゼ移植マウスモデルの両方でISAの測定バイアスを最小限に抑える標準化された方法について説明します。

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), ...

a-standardized-method-for-measuring-internal-lung-surface-area-via

Research

JoVE Journal

Developmental Biology

13.1K ビュー.  Peking University. 内臓の肺表面積（ISA）は、肺疾患および傷害誘発性肺胞再生における肺の形態学および生理学を評価するための決定的な基準である。ここでは、肺の肺切除術とプロテーゼ移植マウスモデルの両方でISAの測定バイアスを最小限に抑える標準化された方法について説明します。 

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

A Simple Method of Mouse Lung Intubation

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

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

マウス肺挿管の簡単な方法

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

医学

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

マウス肺内皮表面層の in vivo測定における

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

肺への癌転移を調査するための永久的な窓

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

がん研究

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 ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

代償肺の成長のマウス肺モデル

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

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 ...

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 ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

腹腔内のブドウ糖負荷試験、肥満や呼吸器アウトカムに対する代謝が異常の影響を研究する肺機能の測定や肺の固定

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

免疫・感染学

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 ...

<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 ...