Name: pseudomonas aeruginosa induced lung injury model
Uploaded: 2014-10-29T15:00:00.000+00:00
Duration: 7 min 24 s
Description: Watch this Scientific Journal Video about Pseudomonas aeruginosa Induced Lung Injury Model at JoVE.com

This work was supported by National Institutes of Health grants HL105947-01 (YL), HL07829-16 (AM), HL090152 (AM).

動物実験は、イリノイ大学シカゴ校の動物ケア委員会と制度バイオセーフティ委員会によって承認された。 注：マスク、目の保護、ガウンやジャンプスーツ、ダブル手袋：シュードモナスを含むすべての手順は、バイオセーフティレベル2（BSL2）が含まれるが、これらに限定されない慣行、で行われるべきである。認定された安全キャビネットで働いています。漂白剤や消毒剤をベース二酸化塩素との細菌に接触して楽器を扱う。輸送サンプルに対して密封されたボックスを使用します。 1. P.緑膿菌文化と成長<ol><li>ストアP. -80℃で凍結バイアルスクリューキャップの細菌株として緑膿菌 PA103。 </li><li> 70％エタノール湿らせたペーパータオルでボックス内のラックにバイアルを置き、バイオセーフティレベル2（BSL2）の研究室に連れて行く。 </li><li>ストリークヒツジ血液寒天プレート上に細菌やインキュベーターで15時間〜37℃で生育する。 </li&gt; <li> BSL2フードでは、5ミリリットルPBSに細菌ループおよび再懸濁したプレートからの細菌を傷つける。 </li><li>最大3ヶ月間、4℃での在庫を保管してください。しかし、2週間ごとに力価を決定。 </li><li>シリアルに（通常は1：10 4〜1時10分〜7）をPBS中の細菌を希釈し、羊血液寒天プレート上の既知の希釈をプレートアウト。 </li><li> 〜15時間プレートをインキュベートします。コロニーをカウントし、細菌濃度を決定するために、コロニー形成単位（CFU）を計算する。 </li><li>適切な濃度を再懸濁（〜5×10 3 CFU /μl）を1.5ミリリットルの滅菌スクリューキャップのクライオバイアル中で0.5mlのPBSで。クリオバイアルを密封し、スナップのふたボックス内の消毒ラディンペーパータオル上クライオバイアルラックに配置します。 </li><li> BSL2動物施設にあるボックスを輸送。かつてそこに、BSL2キャビネットにボックスを開きます。 </li></ol> 2. P.緑膿菌点眼<ol><li>無菌的に生存手術を行う（滅菌手袋、滅菌楽器、および無菌技術）。滅菌（オートクレーブ処理）手術器具のために働いて表面を提供するために滅菌ドレープを使用してください。各気管点滴注入手順の間にホットビーズ滅菌器を使って楽器を殺菌。 </li><li>麻酔の前にマウスの重量を量る。腹腔内に0.2mlのPBS（腹腔内 ） -ケタミン0.1におけるた（100mg / kg）を、キシラジン（5mg / kg）でマウスを麻酔。つま先のピンチに対する非応答性によって麻酔薬の有効性を判断する。麻酔下での乾燥を防ぐために、目に獣医軟膏を使用してください。 </li><li> BSL2キャビネットに外科ボードにマウスを拘束。 </li><li>カットダウンのための領域を識別します。この領域を剃ると3回アルコールとポビドンヨード綿棒を交互に使用して皮膚を準備します。 </li><li>この麻酔は、そのような気管にアクセスするための皮膚切開などの小手術のために十分であるように局所麻酔薬（リドカイン）で切開領域を扱う。 </li><li>首の正中線で小切開（約5mm）を行い、鈍Fを使用優しく気管へのアクセスのために筋肉を動かすためorceps。外科的切開により気管を公開します。 </li><li> 27 Gの針を1ミリリットル使い捨て注射器への菌液を描画します。各マウスについて、適切な濃度（10 5 CFUまでの各マウス）での細菌の20〜30μlの管理。 </li><li>気管内に針を挿入します。気管にゆっくりとソリューションを注入する。 </li><li>通常、その解決策を示している動物のあえぎが、肺に達していることを確認してください。 </li><li>無菌条件下で無菌縫合糸（6-0モノフィラメント）で傷口を閉じます。 </li><li>術後疼痛を制御するため術後鎮痛としては0.1mg / kgを皮下注射によりブプレノルフィンを使用してください。 </li><li>切開の閉鎖に初期麻酔導入から所要時間が15分未満であることを確認してください。 </li><li>注入後、適切なバイオハザードシャープコンテナに注射器や針の処分。 </li><li> DISIベースの二酸化塩素と手術器具を扱うnfectant 15分間、すすぎ、殺菌のために実験室に戻り、必要に応じて再利用します。 </li><li>ハウス暖かい環境できれいなケージで単独でのマウス。 </li><li>それは意識を取り戻し、動き出すまで、動物に30分ごとにチェックします。以降の最初の3日間、手術後12時間の間隔で。 </li><li>実験を通して、動物のBSL2施設でマウスを保管してください。密封されたボックスに含まれている唯一の組織が動物BSL2施設を残していることを確認してください。マウスが瀕死行動のいずれかを表現した場合の研究中の任意の時点で（呼吸困難、嗜眠、穏やかな刺激に応答して歩き回るの失敗として定義される）、頸椎脱臼に続くボトル源からCO 2吸入によりマウスを安楽死させる。 </li><li>麻酔下で放血によって実験対象を安楽死させる。 <ol><li>安楽死させたマウスからの肺組織を収集します。 BSL2フード内でこれらの手順を実行します。必要に応じて、RACに配置された密封されたチューブを使用してサンプルを転送さらにプロセスのスナップ蓋ボックス内の消毒ラディンペーパータオル上Kである。 </li></ol></li></ol>

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The authors have nothing to disclose.

ここで記述するシュードモナスマウス肺損傷モデルは、急性肺損傷または肺炎を以下起こる炎症、肺損傷、修理、および解像度の全体のプロセスを模倣。それは、臨床的に関連して比較的安全で、取り扱いが容易であるという点でいくつかの他の損傷モデルと比較独自の利点を有している。 手順における重要なステップは、菌液の注入が非常に遅くする必要があるとい?...

肺は、環境病原体に暴露し、炎症および損傷1-3の影響を受けやすいされている。そのような肺炎または成人呼吸窮迫症候群（ARDS）のような病理学的状態の間、白血球により放出され、病原体だけでなく、炎症性因子は肺胞細胞1-3の傷害や死を誘導する。これは、傷害の病理の研究、ならびに修復のメカニズムを容易にするために、急性肺損傷の動物モデルを開発することが重要である。 現在、ほとんどの人は高酸素性ブレオマイシン誘発マウス肺損傷モデル4を使用しています。しかし、高酸素のメカニズムは、傷害が肺炎またはARDS 5中に発生する最も一般的な肺損傷と同じではありませんが原因。ブレオマイシン誘発性急性損傷は、臨床コンテキスト4はまれである。ここでは、Pの気管内注射を用いてマウスの肺損傷モデルを報告緑膿菌 6,7。このモデルは、臨床的に関連であり、pを模倣肺炎8下記起こるrocesses。 免疫無防備状態の個体の日和見、院内病原体、Pとして緑膿菌は通常、肺の気道、尿路、火傷、傷に感染し、また他の血液感染症6が発生します 。細菌は病原性因子毒素Aをリリース、6免疫応答を乗算し、トリガします。 P.のイントラtrachael政権緑膿菌、肺炎を引き起こす細菌にヒトへの暴露の状況を反映しており、病理学は、最近報告されたインフルエンザウイルスH1N1誘発性肺傷害モデル9とは異なる可能性が高い。 P.以来緑膿菌は、より病原性の病原体の一部に比べて取り扱いが比較的安全であり、日和見病原体である。私たちはこの方法はと比較して、肺の遠位肺胞領域へより多くの細菌を導入することが観察されるためここでは、細菌を投与するために気管内注入を使用そのような口を介してカテーテルを使用するなど、いくつかの他の手順。 他の急性肺損傷のモデルと比較して、P.ここで説明緑膿菌モデルは、細菌によって、過剰な炎症によって誘発される肺損傷を研究するのに適している。 P.を使用する他の動物モデルとは異なり緑膿菌は、ここでは、ローカライズされた急性肺損傷を誘導するために、これらの細菌の気管内注入を使用して、敗血症10,11を誘導した。

<table><tbody><tr><td>Anesthetic: Ketamin, xylazine, lidocaine, buprenorphine</td><td></td><td></td><td>pharmaceutical grade</td></tr><tr><td>27 G needle</td><td>Fisher</td><td>1482648</td><td></td></tr><tr><td>syringe</td><td>Fisher</td><td>14823434</td><td>1 ml</td></tr><tr><td>scissors</td><td>Fine Science tools</td><td></td><td></td></tr><tr><td>forceps</td><td>Fine Science tools</td><td></td><td></td></tr><tr><td>suture&amp;nbsp;</td><td>Fisher</td><td>19-037-526</td><td></td></tr><tr><td>Eye gauge, glove, gown</td><td></td><td></td><td></td></tr><tr><td>Biosafety Cabinet</td><td></td><td></td><td></td></tr><tr><td>chlorine dioxide based disinfectant</td><td></td><td></td><td>Clidox</td></tr><tr><td>sheep blood agar plates&amp;nbsp;</td><td>Medex supply</td><td>HL-1160</td><td></td></tr></tbody></table>

pseudomonas aeruginosa induced lung injury model

ヒト急性肺損傷及び肺炎を研究するためには、この疾患の多様な病理学的特徴を模倣する動物モデルを開発することが重要である。ここでは、細菌、緑膿菌 （ 緑膿菌またはPA）の気管内注射によるマウス肺損傷モデルを開発した。このモデルを用いて、損傷の初期段階での肺の炎症を示すことができた。また、肺胞の上皮バリア漏出が気管支肺胞洗浄（BAL）を分析することによって観察された。肺胞細胞死が負傷肺から調製した組織を用いたTUNELアッセイにより観察した。損傷後の後期では、我々は、修復プロセスに必要な細胞の増殖を観察した。損傷は7日、P開始から解決した緑膿菌注入 。このモデルは、肺炎の間、肺の炎症、損傷および修復の順次コースを模倣。この臨床的に関連する動物モデルは、fは、修復のメカニズムを病理を研究するのに適している急性肺損傷をollowing、また、この疾患の潜在的治療剤を試験するために用いることができる。

72時間後、P - 24から出発緑膿菌インジェクション 、細胞性の増加は、（ 図1A-D）肺切片において観察された。肺は、96時間後に損傷（ 図1E）から回復し始めた。 7日目にPを投稿する緑膿菌 、正常な肺胞の形態は大部分が（ 図1F）を回復した。 24時間後、P。で調製肺切片を用いたトンネル染色緑膿菌は、肺胞細胞（...

Watch this Scientific Journal Video about Pseudomonas aeruginosa Induced Lung Injury Model at JoVE.com

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

緑膿菌誘発肺傷害モデル

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

pseudomonas-aeruginosa-induced-lung-injury-model

Research

JoVE Journal

Immunology and Infection

Pseudomonas aeruginosa Induced Lung Injury Model

18.4K ビュー.  University of Illinois at Chicago. We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant. 

ビデオ: Pseudomonas aeruginosa Induced Lung Injury Model

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

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

エアロゾル化リポ多糖を使用して生成肺好中球増加のための方法

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

微生物群集を学びます<em&gt;インビボ</em&gt;：ホスト間の媒介の相互作用のモデル<em&gt;カンジダ</em&gt;と<em&gt;緑膿菌</em&gt;航空で

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

免疫担当げっ歯類で堅牢な肺炎モデルはに対して抗菌有効性を評価するために<i&gt; S。ニューモニエ</i&gt;、<i&gt; H。インフルエンザ</i&gt;、<i&gt; K。ニューモニエ</i&gt;、<em&gt; P。緑膿菌</em&gt;または<em&gt; A。バウマニ</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8トランジェントトランスジェニックマウスモデル<em&gt;インビボ</em&gt;炎症反応の長期モニタリング

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

最大急性非致死肺損傷における急性肺損傷の結果のオープン気管切開胃酸吸引マウスモデル

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

医学

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

一方的気管支内酸インスティレーションによる自己限定急性肺損傷のモデル

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

慢性緑膿菌創傷感染の遅延接種モデル

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. アフルギノーサ 抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

直接気管内リポ多糖刺激によるマウスの急性肺損傷の誘導

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

緑膿菌誘発肺傷害モデル

要約

さらに動画を探す

この動画の章

長期的な慢性

エアロゾル化リポ多糖を使用して生成肺好中球増加のための方法

微生物群集を学びます

免疫担当げっ歯類で堅牢な肺炎モデルはに対して抗菌有効性を評価するために

IL-8トランジェントトランスジェニックマウスモデル

最大急性非致死肺損傷における急性肺損傷の結果のオープン気管切開胃酸吸引マウスモデル

一方的気管支内酸インスティレーションによる自己限定急性肺損傷のモデル

慢性緑膿菌創傷感染の遅延接種モデル

P. アフルギノーサ抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

直接気管内リポ多糖刺激によるマウスの急性肺損傷の誘導

緑膿菌誘発肺傷害モデル

要約

さらに動画を探す

この動画の章

長期的な慢性

エアロゾル化リポ多糖を使用して生成肺好中球増加のための方法

微生物群集を学びます

免疫担当げっ歯類で堅牢な肺炎モデルはに対して抗菌有効性を評価するために

IL-8トランジェントトランスジェニックマウスモデル

最大急性非致死肺損傷における急性肺損傷の結果のオープン気管切開胃酸吸引マウスモデル

一方的気管支内酸インスティレーションによる自己限定急性肺損傷のモデル

慢性緑膿菌創傷感染の遅延接種モデル

P. アフルギノーサ 抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー

直接気管内リポ多糖刺激によるマウスの急性肺損傷の誘導

P. アフルギノーサ抗感染性の前臨床評価のための空気・液体界面における気管支上皮細胞とマクロファージの感染3Dコカルチャー