وقد تم تمويل البحوث في مختبر Bragonzi من قبل مؤسسة الإيطالية التليف الكيسي (CFaCore) والاتحاد الأوروبي وF7-2009-223670. تم تنفيذ جزء من هذا العمل في الإنبيق، مختبر المجهر المتقدمة، وأجري التشريح المرضي الماوس في وحدة من تشريح المرضي (معهد سان رافاييل العلمي).

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	<li>Gibson, R., Burns, J. L., Ramsey, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+and+management+of+pulmonary+infections+in+cystic+fibrosis.">Pathophysiology and management of pulmonary infections in cystic fibrosis.</a> Am. J. Respir. Crit. Care. 168, 918-951 (2003).</li><li>Bragonzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+acute+and+chronic+lung+infection+with+cystic+fibrosis+pathogens.">Murine models of acute and chronic lung infection with cystic fibrosis pathogens.</a> IJMM. 300, 584-593 (2010).</li><li>Cash, H. A., McCullough, B., Johanson, W. G., Bass, J. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+chronic+Pseudomonas+aeruginosa+lung+infection.">Murine models of chronic Pseudomonas aeruginosa lung infection.</a> Lab. Anim. 36, 291-312 (2002).</li><li>Bragonzi, 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=Pseudomonas+aeruginosa+microevolution+during+cystic+fibrosis+lung+infection+establishes+clones+with+adapted+virulence+AJRCCM.">Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence AJRCCM.</a> 180, 138-145 (2009).</li><li>Kukavica-Ibrulj, I., Facchini, M., Cigana, C., Levesque, R. C., Bragonzi, A., Filloux, S., Ramos, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Pseudomonas+aeruginosa+virulence+and+the+host+response+using+murine+models+of+acute+and+chronic+lung+infection.">Assessing Pseudomonas aeruginosa virulence and the host response using murine models of acute and chronic lung infection.</a> Methods in Pseudomonas aeruginosa: Humana Press. , (2014).</li><li>Moalli, F., 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=The+Therapeutic+Potential+of+the+Humoral+Pattern+Recognition+Molecule+PTX3+in+Chronic+Lung+Infection+Caused+by+Pseudomonas+aeruginosa.">The Therapeutic Potential of the Humoral Pattern Recognition Molecule PTX3 in Chronic Lung Infection Caused by Pseudomonas aeruginosa.</a> J. Immunol. 186, 5425-5534 .</li><li>Paroni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+CFTR-deficient+mice+to+long-term+Pseudomonas+aeruginosa+chronic+infection+and+PTX3+therapeutic+treatment.">Response of CFTR-deficient mice to long-term Pseudomonas aeruginosa chronic infection and PTX3 therapeutic treatment.</a> J. Infect. Dis. In press, .</li><li>Maxeiner, J., Karwot, R., Hausding, M., Sauer, K. A., Scholtes, P., Finotto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+enable+the+investigation+of+murine+bronchial+immune+cells,+their+cytokines+and+mediators.">A method to enable the investigation of murine bronchial immune cells, their cytokines and mediators.</a> Nat. Protoc. 2, 105-112 (2007).</li><li>Bragonzi, 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=Pseudomonas+aeruginosa+microevolution+during+cystic+fibrosis+lung+infection+establishes+clones+with+adapted+virulence..">Pseudomonas aeruginosa microevolution during cystic fibrosis lung infection establishes clones with adapted virulence..</a> AJRCCM. In press, (2009).</li><li>Pirone, 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=Burkholderia+cenocepacia+strains+isolated+from+cystic+fibrosis+patients+are+apparently+more+invasive+and+more+virulent+than+rhizosphere+strains.">Burkholderia cenocepacia strains isolated from cystic fibrosis patients are apparently more invasive and more virulent than rhizosphere strains.</a> Environ. Microbiol. 10, 2773-2784 (2008).</li><li>Bragonzi, 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=Modelling+co-infection+of+the+cystic+fibrosis+lung+by+Pseudomonas+aeruginosa+and+Burkholderia+cenocepacia+reveals+influences+on+biofilm+formation+and+host+response.">Modelling co-infection of the cystic fibrosis lung by Pseudomonas aeruginosa and Burkholderia cenocepacia reveals influences on biofilm formation and host response.</a> PLoS One. 7, .</li><li>Bianconi, I., 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=Positive+signature-tagged+mutagenesis+in+Pseudomonas+aeruginosa:+tracking+patho-adaptive+mutations+promoting+long-term+airways+chronic+infection.">Positive signature-tagged mutagenesis in Pseudomonas aeruginosa: tracking patho-adaptive mutations promoting long-term airways chronic infection.</a> PLoS Pathog.. 7, (2011).</li></ol>

يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

الخطوات الحاسمة في P. وذكرت الزنجارية حبات إعداد والتحدي الماوس أدناه. سلالة الزنجارية P. المستخدمة للفئران التحدي أمر بالغ الأهمية. الوفيات، عدوى مزمنة أو التخليص ربما تختلف اختلافا كبيرا تبعا لإجهاد البكتيريا تس?...

التليف الكيسي (CF) هو مرض وراثي التي تسببها طفرات في التليف الكيسي عبر الغشاء تصرف منظم (CFTR) الجينات. هذا الجين يشفر لقناة كلوريد أعرب على الغشاء معظم الخلايا الظهارية. تسبب توسع القصبات، يسد المخاط ومتني الدمار أساسا من التهابات الزائفة الزنجارية يؤدي تدريجيا إلى أمراض الرئة الحادة والوفيات في معظم المرضى CF 1. فهم المرضية CF ومزيد من علاجات جديدة تعتمد على تطوير نموذج حيواني مع السمات المميزة لCF. العديد من الفئران، المعدلة وراثيا لهذا الجين CFTR، تم إنشاؤه لكن القيود في قدرة تلك الأنواع ألخص بأمراض الرئة CF-مثل وعدة تشوهات الجهاز التي شوهدت في مرضى التليف الكيسي قد تم توثيقها على نطاق واسع 2. تطوير عدوى هي واحدة من التحديات الرئيسية في نموذج حيواني CF. على البنود الأدبيقترح في وقت مبكر أن عدوى مزمنة دائم أكثر من شهر واحد يمكن أن يتحقق إلا إذا تم تلقيح الفئران مع البكتيريا جزءا لا يتجزأ من وكيل شل حركة مثل أجار، الاغاروز، أو الأعشاب البحرية الجينات 3-5. توفر هذه العوامل شل حركة الشروط microaerobic / اللاهوائية التي تسمح للبكتيريا أن تنمو في شكل microcolonies، على غرار النمو في المخاط من المرضى CF 6. هذا النموذج من عدوى مزمنة تؤدي إلى استمرار البكتيريا في الرئتين تسبب التهاب الشعب الهوائية والأضرار 7. ومع ذلك، اعتمادا على الطريقة المستخدمة، السلالة البكتيرية وجرعة تلقيح في الرئتين، فإن النسبة المئوية من الفئران المصابة المزمن والحمل البكتيري تعافى في الرئتين عند نقاط زمنية مختلفة يمكن أن تختلف إلى حد كبير. على وجه الخصوص، لا يزال التحدي الرئيسي للعدوى مزمنة طويلة الأجل عبء البكتيرية منخفضة من قبل P. الزنجارية ونسبة منخفضة من الفئران المصابة أسابيع بعد التحدي، مشيرا ثايتم مسح الخلايا البكتيرية ر تدريجي من قبل المضيف. عن طريق تحديد P. الزنجارية RP73 سلالة السريرية من مجموعة من CF يعزل 8 حصلنا عليها بنجاح وفيات منخفض، والآفات أكثر حدة، ونسبة عالية من العدوى المزمنة مع الحمولة الجرثومية مستقرة تصل إلى شهر واحد في الفئران C57Bl/6NCrl. تفاصيل هذه الورقة منهجية لتضمين P. الزنجارية في حبات أجار، ولدينا إصابة الفئران عن طريق التقطير داخل القصبة الهوائية، ويقاس الحمل والسيتوكينات البكتيرية في الرئتين، وجمع السائل BAL وأجرى الفحص النسيجي. عموما، فإن هذا البروتوكول مساعدة الباحثين في معالجة المسائل الهامة أساسا على 8،9 المرضية واختبار علاجات جديدة ضد P. الزنجارية عدوى مزمنة 10،11.

<table><tbody><tr><td>Bacto Tryptic Soy Broth</td><td>Becton Dickinson</td><td>211823</td><td></td></tr><tr><td>Difco Agar, granulated</td><td>Becton Dickinson</td><td>214510</td><td></td></tr><tr><td>Heavy mineral oil</td><td>Sigma-Aldrich</td><td>330760-1L</td><td></td></tr><tr><td>S-(+)-Ketamine hydrochloride</td><td>Sigma-Aldrich</td><td>K1884</td><td></td></tr><tr><td>Xylazine hydrochloride</td><td>Sigma-Aldrich</td><td>X1251</td><td></td></tr><tr><td>1 ml Syringe 25 G 5/8 in&amp;nbsp;0.5 mm x 16 mm</td><td>PIC</td><td>3071250300350</td><td></td></tr><tr><td>Catheter 22 G 0.9 mm x 25 mm</td><td>Becton Dickinson</td><td>381223</td><td></td></tr><tr><td>Graefe Forceps - 0.5 mm Tips Curved</td><td>Fine Science Tools</td><td>11152-10</td><td></td></tr><tr><td>Scissors, Iris, 11 cm, straight</td><td>World Precision Instruments</td><td>501758</td><td></td></tr><tr><td>Suture clips</td><td>Fine Science Tools</td><td>12040-01</td><td></td></tr><tr><td>Suture thread</td><td>Fine Science Tools</td><td>18020-40</td><td></td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>BE12-167F</td><td></td></tr><tr><td>Complete protease inhibitor cocktail</td><td>Roche</td><td>11836145001</td><td></td></tr><tr><td>Fast-Read 102 Burker disposable chamber</td><td>Biosigma</td><td>390497</td><td></td></tr><tr><td>Tuerk solution</td><td>Fluka</td><td>93770</td><td></td></tr><tr><td>RBC lysis buffer</td><td>Biolegend</td><td>420301</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Lonza</td><td>DE14-801F</td><td></td></tr><tr><td>EZ cytofunnel</td><td>Thermo Scientific</td><td>A78710021</td><td></td></tr><tr><td>Superfrost ultra plus microscope slides</td><td>Thermo Scientific</td><td>J3800AMNZ</td><td></td></tr><tr><td>Diff-Quik Romanowsky staining set</td><td>Medion Diagnostics</td><td>130832</td><td></td></tr><tr><td>Hexadecyltrimethylammonium chloride</td><td>Sigma-Aldrich</td><td>52366-10G</td><td></td></tr><tr><td>96-well EIA/RIA plate</td><td>Costar</td><td>3590</td><td></td></tr><tr><td>3,3&amp;rsquo;,5,5&amp;rsquo;- Tetramethylbenzidine</td><td>Sigma-Aldrich</td><td>T8665-1L</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>320501-1L</td><td></td></tr><tr><td>10% Neutral buffered formalin</td><td>Bio-Optica</td><td>05-01005Q</td><td></td></tr><tr><td>Harris hematoxylin non Papanicolau</td><td>Bio-Optica</td><td>05-M06004</td><td></td></tr><tr><td>Eosin plus alcoholic solution</td><td>Bio-Optica</td><td>05-M11007</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Shaking incubator</td><td>Amerex Instruments</td><td>Steady Shake 757</td><td></td></tr><tr><td>Water bath</td><td>Grant</td><td>SUB14</td><td></td></tr><tr><td>Homogenizer</td><td>Ystral</td><td></td><td></td></tr><tr><td>Precision balance</td><td>KERN</td><td>440-47N</td><td></td></tr><tr><td>Cytocentrifuge</td><td>Thermo Scientific</td><td>A78300003</td><td></td></tr><tr><td>Low Cost Heating Pad</td><td>2biol</td><td>LCHP</td><td></td></tr><tr><td>Homogenization probe</td><td>Ystral</td><td>2366925(small)</td><td></td></tr><tr><td>Inverted optical microscope</td><td>Zeiss</td><td>Axioplan2</td><td></td></tr><tr><td>Camera (microscope)</td><td>Zeiss</td><td>Axiocam MRc5</td><td></td></tr><tr><td>Rotary microtome</td><td>Leica</td><td>RM2255</td><td></td></tr></tbody></table>

long term chronic pseudomonas aeruginosa airway infection in mice

نموذج الفأر من عدوى المسالك الهوائية المزمن هو أحد الأصول الرئيسية في التليف الكيسي (CF) البحوث، وعلى الرغم من أن هناك عدد من المخاوف بشأن النموذج نفسه. المراحل المبكرة من الالتهاب والعدوى وقد تم دراسة على نطاق واسع باستخدام الزائفة الزنجارية نموذج أجار الخرز الماوس، في حين ركزت تقارير قليلة فقط على العدوى المزمنة على المدى الطويل في الجسم الحي. يبقى التحدي الرئيسي لعدوى مزمنة على المدى الطويل عبء البكتيرية منخفضة من قبل P. الزنجارية ونسبة منخفضة من الفئران المصابة أسابيع بعد التحدي، مشيرا إلى أن الخلايا البكتيرية يتم مسح تدريجيا من قبل المضيف. تعرض هذه الورقة طريقة للحصول على كفاءة عدوى مزمنة طويلة الأجل في الفئران. ويستند هذا الأسلوب على التضمين من P. الزنجارية سلالات السريرية في حبات أجار في المختبر، تليها التقطير داخل القصبة الهوائية في الفئران C57Bl/6NCrl. ويرتبط التهاب في الرئتين الثنائية مع بغازياألطراف قابلة للقياس للقراءة عموميات بما في ذلك فقدان الوزن، وفيات، والعدوى المزمنة، والاستجابة للالتهابات. وP. كان يفضل الزنجارية RP73 سلالة السريرية على مدى سلالة المختبر المرجعي PAO1 لأنه أدى إلى وفيات أقل نسبيا، أشد الآفات، والعدوى المزمنة أعلى. P. قد تستمر الزنجارية الاستعمار في الرئة لأكثر من ثلاثة أشهر. الفئران أمراض الرئة يشبه ذلك من مرضى التليف الكيسي يعانون من أمراض رئوية مزمنة متقدمة. هذا نموذج الفئران يحاكي معظم كثب مسار المرض الإنسان، ويمكن استخدامها على حد سواء لدراسات عن المرضية وتقييم علاجات جديدة.

عندما يتم البروتوكول بشكل صحيح، P. سوف الزنجارية أجار حبات قياس بين 100-200 ميكرون ويمكن ملاحظتها مع ضوء المجهر المقلوب من قبل pipetting كمية صغيرة من حبات أجار التعليق على شريحة. الخلايا البكتيرية واحدة واضحة في حبات أجار، كما هو مبين بالتفصيل في الشكل 1. </...

Watch this Scientific Journal Video about Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice at JoVE.com

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

نحن تصف طريقة أجار حبات لإقامة عدوى مجرى الهواء المستمر على المدى الطويل المزمن الزائفة الزنجارية في نموذج الفأر.

على المدى الطويل المزمن<em&gt; الزائفة الزنجارية</em&gt; عدوى مجرى الهواء في الفئران

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 ...

long-term-chronic-pseudomonas-aeruginosa-airway-infection-in-mice

Research

JoVE Journal

Immunology and Infection

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

22.4K Views.  San Raffaele Scientific Institute.  نحن تصف طريقة أجار حبات لإقامة عدوى مجرى الهواء المستمر على المدى الطويل المزمن الزائفة الزنجارية في نموذج الفأر. 

Video: Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Pseudomonas aeruginosa Induced Lung Injury Model

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. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

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.

الزائفة الزنجارية المستحثة الرئة الإصابات نموذج

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. ...

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.

دراسة المجتمعات الميكروبية<em&gt; في فيفو</em&gt;: نموذج من التفاعل بوساطة بين المضيف<em&gt; المبيضات البيض</em&gt; و<em&gt; الزائفة الزنجارية</em&gt; في الخطوط الجوية

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

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

مزمن السالمونيلا نموذج الفأر المصاب

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Biology

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

والطريقة غير الغازية ومن الناحية الفنية غير كثيفة للتحريض وPhenotyping التجريبية البكتيرية والالتهاب الرئوي في الفئران

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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.

و إيل-8 عابر نموذج الماوس المعدلة وراثيا ل<em&gt; في فيفو</em&gt; رصد طويل الأمد للردود الالتهابية

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

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

البلعمه البلاعم السنخية وإزالة البكتيريا في الفئران

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

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

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

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

جيل طراز انسداد الشعب الهوائية المزمن في الفئران بالتعرض للأوزون المتكررة

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

Medicine

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.

نموذج التلقيح المتأخر لعدوى الجروح المزمنة الزائفة الجيروجينوسا

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

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

نموذج فأر لانتقال العقدية الرئوية من المستعمر إلى العامل الممرض عند العدوى الفيروسية المشتركة يلخص المرض المتفاقم حسب العمر

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

نموذج تجريبي لتقييم حل الالتهاب الرئوي

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

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