Name: intratracheal inoculation of fischer 344 rats with francisella tularensis
Uploaded: 2017-09-30T14:00:00.000+00:00
Duration: 6 min 9 s
Description: Watch this Scientific Journal Video about Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis at JoVE.com

Bu çalışmada sözleşmeli HDTRA1-14-C-0116 savunma tehdit azaltma Ajansı (DTRA) ve enfeksiyon genomik (DOD #W911NF-11-1-0136) mükemmellik merkezi tarafından desteklenmiştir.

Bu iş için bakım ve kullanım laboratuvar hayvanları Ulusal Sağlık Enstitüleri Kılavuzu'nda bulunan öneriler ile sıkı uygun olarak gerçekleştirildi. Kemirgenler içeren hayvan iletişim kuralları onayladı tarafından University of Texas San Antonio kurumsal hayvan bakım ve kullanım Komitesi (IACUC) iletişim kuralı'nı MU009(RA).
 1. kateter hazırlamak, Trakea göstergesi ve F. tularensis Inoculum <ol><li>Hazırlamak kateter (20 G x 2 inç) <ol><li>20 G x 2 iğne ve künt ve düz iğne ucu bitirmek ile eziyet kesilmiş bir döner aracı. 
		Not: Körelmenin iğne uzunluğu yaklaşık 3 mm zaman tamamen körelmenin iğne oturmuş kateter kol içine iptal edin metal uç izin vermelisiniz. Körelmenin iğne kateter yapısal sertlik Trakea kateter yerleştirme olanak verir ve kateter kol çıkıntılı körelmenin iğne ucu yokluğu nefes borusu yaralanma engeller.</li>
		<li>Körelmenin iğneler ve % 70 etanol kullanarak Kateterler temiz ve 15 dakika süreyle UV ışık altında sterilize</li>
	</ol></li> <li>Hazırlamak Trakea göstergesi <ol><li>bir kateter açılış içine oturup bir hava geçirmez mühür formu ipucu izin vermek için 1.000 µL pipet ucu Trim.</li>
		<li>'İ 200 µL aerosol bariyer pipet ucu ve önceden kesilmiş 1.000 µL pipet ucu yerde bir filtreyi kaldırmak.</li>
		<li>Filtre taşıyabilirsiniz serbestçe 1.000 µL pipet ucu içinde belgili tanımlık uç uç aşağı işaretlendiğinde olun.</li>
	</ol></li> <li>F. tularensis Inoculum <ol><li>Grow F. tularensis yük gecede uygun agar plaka 37 ° C. Scrape yaklaşık 100 µL agar plaka ile steril bir bakteriyel çim aşı döngü ve 500 mL Erlenmeyer uygun sıvı büyüme ortamının 250 mL içeren flask aşılamak için kullanın (için Fn: %0,1 L-sistein hidroklorür monohidrat ile desteklenmiş tryptic soya suyu (TSB)) ve gecede 37, sallayarak kuluçkaya ° C. 
		Not: video kullanılan F. novicida %0,1 L-sistein hidroklorür monohidrat ile desteklenmiş bir tryptic soya agar (TSA) plaka üzerinde büyüdü.</li>
		<li>4,221 x g, oda sıcaklığında 10 dakika için de gecede yetişkin sıvı kültür santrifüj kapasitesi ve süpernatant bakteri Pelet aksatmadan kaldırın. 
		Uyarı: Lütfen kurum izleyin &#39; süpernatant atmak için s Biyogüvenlik önerileri.</li>
		<li>Yavaşça bakteriyel Pelet uygun sıvı orta 25 mL ile yeniden askıya alma (Fn: %0,1 L-sistein hidroklorür monohidrat ile desteklenmiş TSB) % 10 gliserol, 25 mL pipet kullanarak içeren.</li>
		<li>1 mL pipet, aliquot tüpleri, 500 µL aliquots içine resuspended bakteri kültürü kullanarak bir kuru buz/etanol banyosunda donma ve depolamak-80 ° C.</li>
		Donmuş kültür tüpleri titresi belirlemek için <li>-80 ° C'de depolanan iki donmuş şişeleri Kaldır, çözülme buzda ve seri dilutions içinde fosfat tamponlu tuz (kaplama tarafından uygun büyüme medyada takip PBS) gerçekleştirmek (Fn: %0,1 içeren TSA L-sistein hidroklorid). 37 ° C'de inkübe 24-48 h tabaklar ve koloni oluşturan birimler (CFU) bakteri hücreleri içinde donmuş kültür tüpleri (iki şişe ortalaması) sayısını hesaplamak için numaralandırılır.</li>
		<li>Bakteriyel inoculum donmuş bir şişe buz çözdürme tarafından hazırlamak ve PBS ile Kültür 10 7 CFU/100 µL son konsantrasyonu oranında seyreltin. İnoculum 100 µL istenen CFU ortaya çıkarır bir konsantrasyon olmalıdır (gliserol inoculum içinde son konsantrasyonu fazla % 3, farenin boğulma önlemek için olamaz). Donmuş kültür tüpleri-80 ° C'de depolanan-ebilmek var olmak kullanılmış için altı ay sonra hazırlık.</li>
	</ol></li></ol> 2. Sıçan anestezi <ol><li>anestezi odası bir anestezi makinesi uygun şekilde çalışan isoflurane vaporizatör için bağlanmak.</li>
	<li>Tüp gaz sistemi atma anestezi odasına atma gaz bağlayın.</li>
	<li>Oksijen akışı 4 L/dak için anestezi makinede açın</li>
	<li>% 5 isoflurane Buharlaştırıcı ayarla.</li>
	<li>Anestezi odasında fare yerleştirin. 
	Not: Biz genellikle kullanmak fareler arasında 8-10 hafta eski (130-180 g); Genç sıçanlar ağız boşluğuna küçük boyutu nedeniyle bu tekniği ile aşılamak zor.</li>
	Anestezi indüksiyon, gerçekleştiğinde <li>korumak %5 isoflurane, sıçan, 2 L/dak oksijen etkisi. Bu yaklaşık 3-10 dakika sürer</li>
	<li>Kalite ve solunum ve kalp atışı ve refleks uyarım ayak çimdik testi olduğu gibi tepki oranı ile anestezi derinliği belirler. İdeal anestezi derinliği 1-1.5 s sayım her nefes arasında belirttiği.</li>
</ol> 3. Boğaza aşılama <ol><li>fare anestezi odasından çıkarın ve sıçan dorsally kemirgen entübasyon kürsüye yerleştirin. Sıçan tutmak için tutucu sıçan ön dişleri iliştirin. 
	Not: sıçan işlem sırasında uyandırmak başlarsa, kateter-ebilmek var olmak çıkarmak ve fare anestezi daha derin bir uçak ulaşmak için anestezi odasına döndü.</li>
	<li>Dil desteği el olarak aynı tarafında geniş nokta soyunma başparmak Forseps ile hareket için dominant el kullanın.</li>
	<li>Kullanım desteklemek el ve dil laringoskop karşı fare ile güvenli &#39; s alt çene ve trakea ve yemek borusu hayvan görselleştirin. Nefes borusu açık ve fare nefes alır gibi yakın.</li>
	Bir 20'lik blunted-iğne, içeren kateter <li>Ekle hazırlanan adım 1.1, nefes borusu içine. Hafif direnç olabilir ve nefes borusu içine ekleme olabilir &#34; inişli çıkışlı &#34; trakeal kıkırdak sürtünme kateter nedeniyle. Nem veya sığ solunum nedeniyle, Trakea Trakea görselleştirme önleyen gırtlak kapağını tarafından karşılanabilir. Yavaşça gırtlak kapağını kıkırdak kenarına dokunmak kıkırdak kapağı açın ve nefes borusu ortaya çıkarmak neden olur.</li>
	<li>Kör iğne kateter sağlanması sırasında kateter üzerinden kalır borusunda kaldır.</li>
	<li>İzin için nefes borusu eklenen kateter çevresinde edebilmek iri fare geçmek için birkaç saniye.</li>
	<li>Sıkıca Trakea göstergesi üzerine açılış kateter koltuk. Aerosol bariyer hareket belirtmek kateter borusu eklenen doğru.</li>
	<li>Öyle ki hayvanın göğüs uçak entübasyon stand dik bakan sıçan entübasyon kürsüye yatıyor emin olmak.</li>
	<li>Nefes borusu gösterge kaldırmak ve 100 µL teslim (10 7 CFU) F. tularensis 200 µL pipet ucu kullanarak içeren inoculum sıkıca uç kateter açılış karşı oturma.</li>
	<li>Hava ile dolu bir 1 mL kayma ipucu tüberkülin şırınga ve hava inoculum sıçan akciğerlere ulaşır emin olmak için 300 µL gönderin.</li>
	<li>Kateter sıçan cerrahi perondan sökün trakea ve.</li>
	<li>Uyandırmak ve kafes dönmek fareyi izin. Solunum normale döndü emin olun.</li>
	<li>F. tularensis CFU belirlemek 1.3.5 CFU onaylamak için adımda anlatıldığı gibi inoculum teslim borusunda.</li>
	<li>3.1-3,9 unvaccinated (saf) hayvanlar ile inoculum yerine 100 µL PBS üzerinde adımlarını yineleyin.</li>
</ol>

<ol>
	<li>Ellis, J., Oyston, P. C. F., Green, M., Titball, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tularemia.">Tularemia.</a> Clin Microbiol Rev. 15 (4), 631-646 (2002).</li><li>Lyons, C. R., Wu, T. H. <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+Francisella+tularensis+infection.">Animal models of Francisella tularensis infection.</a> Ann N Y Acad Sci. 1105, (2007).</li><li>Hutt, J. A., Lovchik, J. A., Dekonenko, A., Hahn, A. C., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Natural+History+of+Pneumonic+Tularemia+in+Female+Fischer+344+Rats+after+Inhalational+Exposure+to+Aerosolized+Francisella+tularensis+subspecies+tularensis+Strain+Schu+S4.">The Natural History of Pneumonic Tularemia in Female Fischer 344 Rats after Inhalational Exposure to Aerosolized Francisella tularensis subspecies tularensis Strain Schu S4.</a> Am J Pathol. 187 (2), 252-267 (2017).</li><li>Ray, H. 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=The+Fischer+344+rat+reflects+human+susceptibility+to+Francisella+pulmonary+challenge+and+provides+a+new+platform+for+virulence+and+protection+studies.">The Fischer 344 rat reflects human susceptibility to Francisella pulmonary challenge and provides a new platform for virulence and protection studies.</a> PloS one. 5, e9952 (2010).</li><li>Jemski, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+tularemia:+comparison+of+selected+routes+of+vaccination+in+Fischer+344+rats.">Respiratory tularemia: comparison of selected routes of vaccination in Fischer 344 rats.</a> Infect Immun. 34 (3), 766-772 (1981).</li><li>Wu, T. H., 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=Vaccination+of+Fischer+344+rats+against+pulmonary+infections+by+Francisella+tularensis+type+A+strains.">Vaccination of Fischer 344 rats against pulmonary infections by Francisella tularensis type A strains.</a> Vaccine. 27 (34), 4684-4693 (2009).</li><li>Mara-Koosham, G., Hutt, J. A., Lyons, C. R., Wu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+contribute+to+effective+vaccination+against+respiratory+infection+by+type+A+Francisella+tularensis+strains.">Antibodies contribute to effective vaccination against respiratory infection by type A Francisella tularensis strains.</a> Infect Immun. 79 (4), 1770-1778 (2011).</li><li>Oyston, P. C., Sjostedt, A., Titball, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tularaemia:+bioterrorism+defence+renews+interest+in+Francisella+tularensis.">Tularaemia: bioterrorism defence renews interest in Francisella tularensis.</a> Nat Rev Microbiol. 2 (12), 967-978 (2004).</li><li>Kingry, L. C., Petersen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+review+of+Francisella+tularensis+and+Francisella+novicida.">Comparative review of Francisella tularensis and Francisella novicida.</a> Front Cell Infect Microbiol. 4, 35 (2014).</li><li>Rohmer, 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=Comparison+of+Francisella+tularensis+genomes+reveals+evolutionary+events+associated+with+the+emergence+of+human+pathogenic+strains.">Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains.</a> Genome Biol. 8 (6), R102 (2007).</li><li>Chu, P., 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=Live+attenuated+Francisella+novicida+vaccine+protects+against+Francisella+tularensis+pulmonary+challenge+in+rats+and+non-human+primates.">Live attenuated Francisella novicida vaccine protects against Francisella tularensis pulmonary challenge in rats and non-human primates.</a> PLoS Pathog. 10 (10), e1004439 (2014).</li><li>Signarovitz, A. 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=Mucosal+Immunization+with+Live+Attenuated+Francisella+novicida+U112&#916;iglB+Protects+against+Pulmonary+F.+tularensis+SCHU+S4+in+the+Fischer+344+Rat+Model.">Mucosal Immunization with Live Attenuated Francisella novicida U112&#916;iglB Protects against Pulmonary F. tularensis SCHU S4 in the Fischer 344 Rat Model.</a> PloS one. 7 (10), e47639 (2012).</li><li>Cunningham, A. 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=Enhancement+of+vaccine+efficacy+by+expression+of+a+TLR5+ligand+in+the+defined+live+attenuated+Francisella+tularensis+subsp.+novicida+strain+U112DiglB::fljB.">Enhancement of vaccine efficacy by expression of a TLR5 ligand in the defined live attenuated Francisella tularensis subsp. novicida strain U112DiglB::fljB.</a> Vaccine. 32 (40), 5234-5240 (2014).</li></ol>

Yazarlar ifşa yoktur

Fischer 344 sıçan Tularemi aşısı geliştirme3için önemli bir model haline geliyor. Bunlar aerosoller teslim edildiğinden F. tularensis maruz pulmoner rota üzerinden weaponized F. tularensis, türlü karşı etkinlik gösteren için önemlidir. Farenin boğaza aşılama maruz sıçan akciğerlerinin F. tularensis ekipmanları üreten büyük, pahalı ve karmaşık aerosol gerek kalmadan kolaylaştırır. F. tularensis seçin Aracısı formları Ayrıca k...

F. tularensis (Ft) insan hastalık nedenleri Tularemi. Bakteriler akciğer yönlendirme yolu iktisap zaman, bu yüksek morbidite ve mortalite1öğretilenbir Tularemi için yol açar. F. tularensis aerosolize formlarla ilişkili tehlike nedeniyle bir biothreat ajan olarak kabul edilir ve şu anda ABD'de insan kullanımı için onaylanan aşı Yoğun bir çaba aşı ve bu bakteriyel biothreat insan nüfusu ve yasadışı kullanıma karşı korumak için öğretilenbir Tularemi karşı tedavi edici önlemler geliştirmek için şu sıralar başlatıldı.
Tularemi araştırma çoğunu fareler F. tularensis enfeksiyon için aşırı duyarlılık ve reaktifler yaygınlığı nedeniyle fare modeli üzerinde odaklanmıştır. Ancak, fareler için bu model2aşı etkinlik gösteren zorluğu nedeniyle aşı geliştirme zor bir model olduğu kanıtlanmıştır. Son zamanlarda, Fischer 344 sıçan Tularemi aşısı geliştirme3için bir model olarak geliştirilmiştir. Çeşitli F. tularensis alttür Fischer 344 fare duyarlılığını insan duyarlılığı4taklit eder ve fareler F. tularensis pulmoner meydan karşı korumak için bilinen bir canlı aşı suşu ile aşı tarafından korunabilir insanlar5,6,7. F. tularensis enfeksiyon insanların bazı özellikleri Fischer 344 fare modelleri, pulmoner F. tularensis karşı koruyan bir aşı geliştirilmesi için son derece faydalı bir model olması olabilir maruz kalma.
Etkili bir aşı insanlara F. tularensispulmoner maruz karşı korumak gerekir. Büyük olasılıkla pulmoner maruz kalma weaponized F. tularensis aerosolize bakteriler akciğer8içine inhale olurdu. Bununla birlikte, F. tularensis nesil aerosol tehlikeli ve hantal ve özel ekipman ve koruma gerekir. Alternatif bir yol içinde belki de daha fazla olduğunu sıçan pulmoner maruz kalma uyarlanabilir birden fazla laboratuarlar için özel ekipman eksik boğaza aşı6yolu ile konuşuyor. Laringoskop bir kateter imzalat bir sıçan Trakea içinde doğru yerleşimini için bu tekniği kullanır. Nefes borusu, yemek borusu yerine içinde bulma dan akciğerlere hava akımı görüntüler basit bir aygıt tarafından doğrulanır. F. tularensis daha sonra kateter aracılığıyla ciğerlerine yönetim tarafından bakteri pulmoner teslim edilmesini sağlamak için kateter hava giriş tarafından takip bir şırınga ile teslim edilir. Buna ek olarak, Sıçanlarda etkilerinin bu aşı belirten Jemski,5 içine Fischer 344 fareler burunici rota yolu ile aşılanmış F. tularensis akciğerlerden 3 gün sonrası aşılama kadar kültürlü değil ki daha önce rapor mu değil doğrudan teslim sonuç ciğerlerine bakterilerin.
F. tularensis (F. tularensis subsp. tularensis, F. tularensis subsp. holarctica) SELECT Ajan formları videografisi önleyecek Biyogüvenlik seviye 3 (BSL3) kapsama yordamlar gerektirir. Ancak, F. novicida (Fn) seçin Aracısı durumu nedeniyle onun avirulence sağlıklı insanlarda muaf ve seviye 2 (BSL2) koşulları9,10altında güvenle kullanılabilir. Ayrıca, Fn via boğaza aşılama11,12,13teslim F. tularensis pulmoner pozlama karşı koruyabilirsiniz canlı zayıflatılmış aşılar için temel olarak hizmet vermektedir. Burada sunulan tekniği fareler insanlar için bir model olarak kullanan pulmoner rota üzerinden oluşan enfeksiyonlar incelenmesi için izin verir. Bu teknik özel aerosol üreten ekipman için gerek kalmadan gerçekleştirilebilir. FN burada filme teknikleri için kullanıldı.

<table><tbody><tr><td>GreenLine fiber optic blade size 0</td><td>Carefusion</td><td>5-5231-00</td><td>Macintosh American profile</td></tr><tr><td>GreenLight system laryngoscope handle</td><td>Carefusion</td><td>4559GSP</td><td></td></tr><tr><td>Exel International Safelet I.V. Catheter</td><td>EXEL INTERNATIONAL</td><td>26743</td><td></td></tr><tr><td>Slip Tip Sterile Syringes 1mL</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Broad Point Dressing Thumb Forceps</td><td>Thermo Scientific</td><td>76-302</td><td></td></tr><tr><td>200 &amp;mu;L barrier tip</td><td>GeneseeScientific</td><td>24-142</td><td></td></tr><tr><td>1,000 &amp;mu;L pipette tip</td><td>Olympus Plastics</td><td>24-173</td><td></td></tr><tr><td>Dremel 3000-2/28 Rotary tool kit</td><td>Dremel</td><td>3000228</td><td></td></tr><tr><td>Rodent Intubation Stand</td><td>Braintree Scientific</td><td>RIS 200</td><td></td></tr><tr><td>Isoflurane</td><td>Butler Schein</td><td>NDC 11695-6776-2</td><td></td></tr><tr><td>Rodent anesthesia machine</td><td>Surgivet</td><td>VTC302 Classic T3</td><td></td></tr><tr><td>Rodent Anesthesia chamber</td><td>Braintree Scientific</td><td>AB 1</td><td></td></tr></tbody></table>

intratracheal inoculation of fischer 344 rats with francisella tularensis

Pulmoner enfeksiyon bakteri FRANCISELLA tularensis ile ciddi ve ölümcül hastalık, Tularemi, insanlarda yol açabilir. Bir onaylanmış Tularemi aşısı insanlar için geçerli olmaması nedeniyle araştırma uygun hayvan modelleri kullanan aşı geliştirme üzerine odaklanmıştır. Fischer 344 sıçan F. tularensis enfeksiyon insan duyarlılık yansıtır ve bu nedenle Tularemi aşısı geliştirme için çekici bir model bir model olarak ortaya çıkmıştır. F. tularensis Fischer 344 fareyle boğaza aşı insanlarda akciğer pozlama taklit eder. Sıçan nefes borusu içine başarılı teslimat Akciğer teslimat için önemlidir. Laringoskop aydınlatma ile düzgün imzalat sıçan tracheae entübasyon için kullanılır; Nefes borusu içinde doğru bulma nefes algılamak için basit bir aygıt tarafından belirlenir. Entübasyon, F. tularensis kültürü şırınga ile ölçülen bir doz teslim edilir. Bu teknik aşısı etkinliğini değerlendirmek için fare nefes borusu içinde F. tularensis pulmoner teslim standartlaştıran.

F. tularensis boğaza aşı içinde fare humoral yanıt enzim bağlı immunosorbent assay (ELISA) bakteri, UV-inaktive karşı tarafından belirlenen11daha önce açıklandığı gibi. Toplam immünoglobulin G (IgG) Fischer 344 sıçan Inaktif tüm cep telefonu bakteri öldürülmesinin biçilen post-boğaza aşılama ile Fn bir zayıflatılmış strain (107 CFU inoculum) 14 gün ve gün 28 (şekil 1). Sahte aşı f...

Watch this Scientific Journal Video about Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis at JoVE.com

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Bu iletişim kuralı boğaza aşılamalar Fischer 344 sıçan ile açıklar FRANCISELLA tularensis. Bu yordamı insanlar pulmoner maruz kalma bu potansiyel biothreat Ajan taklit eder ve aşı ve tedavi edici etkinliği pulmoner Tularemi karşı test etmek için kullanılabilir.

Boğaza aşılama, Fischer 344 farelerle FRANCISELLA tularensis

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the ...

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Immunology and Infection

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

7.1K Görüntüleme.  University of Texas San Antonio. Bu iletişim kuralı boğaza aşılamalar Fischer 344 sıçan ile açıklar FRANCISELLA tularensis. Bu yordamı insanlar pulmoner maruz kalma bu potansiyel biothreat Ajan taklit eder ve aşı ve tedavi edici etkinliği pulmoner Tularemi karşı test etmek için kullanılabilir.

Video: Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Akış Sitometri Akciğerler ve Akciğer Boşaltma lenf düğümleri Analizi fareler cerrahi olmayan intratrakeal damlatma

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

İmmünoloji ve Enfeksiyon

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

Noninvaziv Entübasyon Fare Akciğer Patolojisi ve Fizyolojisi Eğitim için

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Tıp

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

Alternatifi olarak dilaltı immünoterapi Akut Solunum Enfeksiyonları Karşı Koruma tetikleyebilecek

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Entübasyon-aracılı intratrakeal (IMIT) damlatma: A Noninvaziv, Lung-özel Dağıtım Sistemi

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

intra-trakeal İdaresi<em&gt; Haemophilus influenza</em&gt; Fare Modelleri Havayolu inflamasyonu Eğitim için

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

Yalıtım için Yöntem Francisella tularensis Dış Membranlar

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

Biyoloji

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.

Immünkompetan Kemirgenler bir Sağlam Pnömoni Modeli karşı antibakteriyel etkinliğini değerlendirmektir<i&gt; S. pneumoniae</i&gt;<i&gt; H. influenza</i&gt;<i&gt; K. pneumoniae</i&gt;<em&gt; P. aeruginosa</em&gt; ya da<em&gt; A. baumannii</em

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

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

İki fotonlu Intravital mikroskobu kullanılarak grip virüsü enfeksiyonu sırasında trakeal mukozasında görüntüleme hücre etkileşimi

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

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.

Doğrudan Intratracheal Lipopolysaccharide Instilasyon ile farelerde akut akciğer hasarının İndükleme

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

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.

Pnömoni Çözünürlüğünü Değerlendirmek için Deneysel Model

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

Boğaza aşılama, Fischer 344 farelerle FRANCISELLA tularensis

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