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).

在动物实验中批准了伊利诺伊大学芝加哥分校的动物护理委员会和机构生物安全委员会。 注意:口罩，保护眼睛，礼服或连身衣，和双层手套:所有涉及假单胞菌程序应与生物安全2级（BSL2）的做法，其中包括但不限于执行。在认证的生物安全柜的工作。与细菌漂白粉或二氧化氯基消毒剂接触治疗手段。用密封箱运输样品。 1. P.铜绿假单胞菌培养与成长<ol><li>商店P.绿脓杆菌 PA103作为细菌的库存在螺旋盖冷冻管在-80℃下。 </li><li>放置在一个架子的小瓶中，用70％乙醇润湿的纸巾的盒子，并采取生物安全等级2（BSL2）实验室。 </li><li>条纹的细菌到绵羊血琼脂平板上并生长在37℃〜15小时，在培养箱中。 </li&gt; <li>在一个BSL2罩，从刮片细菌环和悬浮的细菌，5毫升的PBS。 </li><li>存储库存在4℃下进行长达3个月。然而，确定的效价，每2个星期。 </li><li>串行（1:10 4，通常1:10 7）稀释细菌在PBS和板出来羊血琼脂平板上已知的稀释。 </li><li>孵育板为〜15小时。菌落计数并计算菌落形成单位（CFU），以确定细菌浓度。 </li><li>重悬在合适的浓度（〜5×10 3 CFU /微升）在0.5ml PBS中于1.5ml无菌螺旋盖离心管。密封冷冻管，并放置在消毒上载货纸巾在瞬间盖盒一离心管架。 </li><li>运输箱，以BSL2动物设施。一旦出现，打开了BSL2柜箱。 </li></ol> 2. P.铜绿假单胞菌灌输<ol><li>进行生存无菌手术（无菌手套，无菌器械和无菌技术）。使用无菌的悬垂性，为无菌（蒸压）手术器械提供了工作面。使用消毒每气管滴入程序之间的热珠灭菌仪器。 </li><li>前麻醉小鼠的体重。麻醉小鼠用氯胺酮（100毫克/千克），甲苯噻嗪（5毫克/千克），在0.1 - 0.2毫升的PBS腹膜内（IP）的 。非响应性脚趾捏确定麻醉剂的效力。使用对眼睛的兽医药膏，以防止在麻醉下干燥。 </li><li>抑制小鼠在BSL2柜外科板。 </li><li>识别切割下来的区域。剃此区域，并准备利用交替醇和聚维酮碘拭子3倍的皮肤。 </li><li>治疗与局部麻醉剂（利多卡因），为这个麻醉足以进行小手术如皮肤切口进入气管切口的区域。 </li><li>作一个小切口（约5mm），在颈部的中线，并用钝˚Forceps轻轻移动肌肉访问气管。暴露气管通过手术剥离。 </li><li>吸取菌液到1毫升一次性注射器用27G的针头。对于每个小鼠，给药20-30微升的细菌在适当的浓度（高达10 5 CFU每只小鼠）。 </li><li>将针头插入气管。慢慢注入溶液到气管。 </li><li>确保动物喘气，这通常表示该解决方案已经达到了进入肺部。 </li><li>关闭无菌条件下无菌缝合线（6-0单丝）的伤口。 </li><li>用0.1毫克/千克丁丙诺啡皮下注射作为术后镇痛控制手术后疼痛。 </li><li>确保从初始麻醉诱导至切口闭合所需的时间小于15分钟。 </li><li>注射后，在适当的生物危害的尖锐物容器处置注射器和针头。 </li><li>治疗与二氧化氯基消毒药剂的外科手术器械nfectant 15分钟，冲洗，并返回到实验室进行灭菌，并根据需要重新使用。 </li><li>房子小鼠单独在清洁笼子里的温暖的环境。 </li><li>检查动物每隔30分钟，直到它恢复意识，并开始移动;后来，在12小时的时间间隔为第3天手术后。 </li><li>保持在整个实验动物BSL2设施的小鼠。确保只有包含在密封的盒子组织离开动物BSL2设施。如果小鼠表达任何垂死的行为（定义为呼吸困难，嗜睡，故障响应温柔的刺 ​​激走动），在该研究中的任何一点，从颈椎脱位瓶装源安乐死的小鼠通过CO 2吸入。 </li><li>安乐死放血在麻醉状态下的实验对象。 <ol><li>从安乐死的小鼠收集肺组织。进行在一个BSL2引擎盖这些过程。如果需要，可以使用密封管放入外消旋转移样品钾对载货消毒纸巾在进一步处理易如反掌盖盒。 </li></ol></li></ol>

<ol>
	<li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li><li>Matthay, M. A., Ware, L. B., Zimmerman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li><li>Shimabukuro, D. W., Sawa, T., Gropper, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury+and+repair+in+lung+and+airways.">Injury and repair in lung and airways.</a> Crit Care Med. 31 (8), 524-531 (2003).</li><li>Wansleeben, C., Barkauskas, C. E., Rock, J. R., Hogan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+of+the+adult+lung:+Their+development+and+role+in+homeostasis,+regeneration,+and+disease.">Stem cells of the adult lung: Their development and role in homeostasis, regeneration, and disease.</a> Wiley Interdiscip Rev Dev Biol. 2, 131-148 (2013).</li><li>Singleton, P. A., 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=Dynamin+2+and+c-abl+are+novel+regulators+of+hyperoxia-mediated+nadph+oxidase+activation+and+reactive+oxygen+species+production+in+caveolin-enriched+microdomains+of+the+endothelium.">Dynamin 2 and c-abl are novel regulators of hyperoxia-mediated nadph oxidase activation and reactive oxygen species production in caveolin-enriched microdomains of the endothelium.</a> J Biol Chem. 284, 34964-34975 (2009).</li><li>Sadikot, R. T., Blackwell, T. S., Christman, J. W., Prince, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen-host+interactions+in+pseudomonas+aeruginosa+pneumonia.">Pathogen-host interactions in pseudomonas aeruginosa pneumonia.</a> Am J Respir Crit Care Med. 171, 1209-1223 (2005).</li><li>Liu, Y., 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=Foxm1+mediates+the+progenitor+function+of+type+ii+epithelial+cells+in+repairing+alveolar+injury+induced+by+pseudomonas+aeruginosa.">Foxm1 mediates the progenitor function of type ii epithelial cells in repairing alveolar injury induced by pseudomonas aeruginosa.</a> J Exp Med. 208, 1473-1484 (2011).</li><li>Kurahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+septic+shock+in+pseudomonas+aeruginosa+pneumonia.">Pathogenesis of septic shock in pseudomonas aeruginosa pneumonia.</a> J Clin Invest. 104, 743-750 (1999).</li><li>Kumar, P. A., 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=Distal+airway+stem+cells+yield+alveoli+in+vitro+and+during+lung+regeneration+following+h1n1+influenza+infection.">Distal airway stem cells yield alveoli in vitro and during lung regeneration following h1n1 influenza infection.</a> Cell. 147, 525-538 (2011).</li><li>Delano, M. J., 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=Sepsis+induces+early+alterations+in+innate+immunity+that+impact+mortality+to+secondary+infection.">Sepsis induces early alterations in innate immunity that impact mortality to secondary infection.</a> J Immunology. 186, 195-202 (2011).</li><li>Lange, M., 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=A+murine+model+of+sepsis+following+smoke+inhalation+injury.">A murine model of sepsis following smoke inhalation injury.</a> Biochem Biophys Res Commun. 391 (3), 1555-1560 (2010).</li><li>Kobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+chemokines+in+neutrophil+biology.">The role of chemokines in neutrophil biology.</a> Front Biosci. 13, 2400-2407 (2008).</li><li>Matute-Bello, G., 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=Animal+models+of+acute+lung+injury.">Animal models of acute lung injury.</a> Am J Physiol Lung Cell Mol Physiol. 295, L379-L399 (2008).</li><li>Sadikot, R. T., 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=Targeted+immunomodulation+of+the+nf-kappab+pathway+in+airway+epithelium+impacts+host+defense+against+pseudomonas+aeruginosa.">Targeted immunomodulation of the nf-kappab pathway in airway epithelium impacts host defense against pseudomonas aeruginosa.</a> J Immunol. 176, 4923-4930 (2006).</li><li>Pinto, I. L., 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=Development+of+an+experimental+model+of+neutrophilic+pulmonary+response+induction+in+mice.">Development of an experimental model of neutrophilic pulmonary response induction in mice.</a> J Pneumologia. 29, 213-214 (2003).</li></ol>

The authors have nothing to disclose.

我们在这里描述的假小鼠肺损伤模型模拟炎症，肺损伤，修复和解决发生的急性肺损伤或肺炎的全过程。它具有与其他几个损伤模型中，这是临床相关的和相对安全的和易于处理的比较独特的优势。 在该过程的关键步骤是，在注射细菌溶液必须是很慢的。如果注射过快，老鼠有可能被噎到死。一个成功的注射后，老鼠通常会显示几个深呼吸和喘息，这将有助于微粒进入肺部?...

肺部暴露于环境中的病原体，并很容易受到炎症和损伤1-3。在病理条件下，如肺炎或成人呼吸窘迫综合征（ARDS），病原体以及由白细胞释放炎性细胞因子诱导损伤的肺泡细胞1-3和死亡。它发展为急性肺损伤的动物模型，以促进损伤的病理修复的研究以及机制是很重要的。 目前，大多数人使用高氧和博来霉素诱导的小鼠肺损伤模型4。然而，氧的机制引起的损伤是不一样的肺炎或急性呼吸窘迫综合征5过程中出现的最常见的肺损伤。博莱霉素诱导的急性损伤是罕见的在临床方面4。在这里，我们报告使用气管内注射P的小鼠肺损伤模型铜绿假单胞菌 6,7。这个模型是临床上相关的，并且模仿在p这种情况发生以下肺炎8 rocesses。 由于免疫功能低下的人的机会，院内病原体，P.假单胞菌通常感染肺部，尿路，烧伤，创伤，并且还引起其他血液感染6。细菌释放出毒力因子外毒素A，乘，引发免疫反应6。 P的内部trachael管理假单胞菌反映到引起肺炎的细菌的情况，人体暴露和病理学可能是从最近报道流感病毒H1N1引起的肺损伤模型9不同。由于P.铜绿假单胞菌是一种条件致病菌，它是相对安全的处理相比，一些更致命的病原体。在这里，我们使用气管内注射给药的细菌，因为我们观察到，该方法引入更多的细菌进入肺的较远端肺泡区一些其他方法，例如通过口用的导管。 与其他急性肺损伤模型相比，P。这里描述绿脓杆菌模型适用于研究肺损伤的细菌和过度炎症反应。与使用P。等的动物模型铜绿假单胞菌诱发败血症10,11，这里我们使用气管内注入这些细菌诱发局部急性肺损伤。

<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

为了研究人的急性肺损伤和肺炎，它开发的动物模型来模拟这种疾病的各种病理特征是重要的。在这里，我们已经开发出通过气管内注射的细菌绿脓杆菌 （ 铜绿假单胞菌或PA）的小鼠肺损伤模型。利用该模型，我们能够显示肺部炎症损伤的早期阶段。此外，肺泡上皮屏障泄漏观察到通过分析支气管肺泡灌洗液（BAL）;并且观察到通过使用从受伤的肺组织制备TUNEL测定肺泡细胞死亡。在受伤后的稍后阶段，我们观察到所需的修复过程的细胞增殖。伤病得到了解决，从体育的开始7天后铜绿假单胞菌注射液。这个模型模拟的肺部炎症，损伤和修复的连续过程中的肺炎。这与临床相关的动物模型是适用于研究病理，修复机制中，following急性肺损伤，并且还可以用来测试潜在的治疗剂，这种疾病。

72小时后P. -从24日开始假单胞菌注射，增加的细胞结构中观察到的肺切片（ 图1A-D）。肺开始从96小时后的损伤（ 图1E）中恢复。 7天后P.铜绿假单胞菌 ，正常肺泡形态基本恢复（ 图1F）。隧道染色用肺切片在24小时后的第制备假单胞菌表明细胞死亡中的肺泡细胞（ 图1G-1）。为了研究这种损伤模型中的修复过程中?...

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.

绿脓杆菌引起的肺损伤模型

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 次观看.  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. 

视频: 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.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

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 ...

科研

免疫与感染

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.

采用雾化脂多糖一种产生肺中性粒细胞增多

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.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

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.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</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瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

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.

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

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.

单方支气管内酸灌输自我限制急性肺损伤模型

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.

慢性假单位病伤口感染的延迟接种模型

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. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

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.

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

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

绿脓杆菌引起的肺损伤模型

摘要

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此视频中的章节

长期慢性

采用雾化脂多糖一种产生肺中性粒细胞增多

研究微生物群落

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

IL-8瞬时转化的小鼠模型

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

单方支气管内酸灌输自我限制急性肺损伤模型

慢性假单位病伤口感染的延迟接种模型

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

绿脓杆菌引起的肺损伤模型

摘要

探索更多视频

此视频中的章节

长期慢性

采用雾化脂多糖一种产生肺中性粒细胞增多

研究微生物群落

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

IL-8瞬时转化的小鼠模型

急性肺损伤开放气管切开胃酸吸入小鼠模型结果在急性最大非致命性肺损伤

单方支气管内酸灌输自我限制急性肺损伤模型

慢性假单位病伤口感染的延迟接种模型

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

通过直接内切内脂多糖灌输诱导小鼠急性肺损伤

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估