Gli autori ringraziano Brian Johnson del Istologia ed Imaging Nucleo presso l&#39;Università di Washington per aiutare con la colorazione tricromica e l&#39;analisi. Questo lavoro è stato sostenuto da NIH sovvenzioni HL098067 e HL089455.

L&#39;animale cura e l&#39;uso comitati istituzionali (IACUC) presso l&#39;Università di Washington e Cedars-Sinai Medical Center hanno approvato il lavoro degli animali necessari per questi studi. 1. Preparazione <ol><li> Sterilizzare entrambe le pinze punta smussata e il divaricatore tramite autoclave. </li><li> Usando una cappa di sicurezza biologica, preparare uno stock di lavoro di BLM in PBS dalla polvere liofilizzata. Sonicare la soluzione per 10 minuti a 35 Khz per assicurare una miscelazione uniforme. Nota: Si raccomanda un volume totale di tra 30 e 45 ml per evitare variazioni pipettaggio sulla fascia bassa, e il soffocamento con volumi maggiori. </li><li> Preparare un lavoro pulito che comprende circa 1 m 2 per la procedura stessa, così come luoghi designati per gabbie sia prima che dopo la procedura. </li><li> Fissare la base della tavola procedimento al banco immediatamente davanti ricercatore ponendo 2 o 3 strisce di nastro laboratorio attraverso la base e sottocaricoying panchina. Vedere la Figura 1 per ulteriori specifiche sulla creazione di un bordo. </li><li> Legare una sola lunghezza di dimensioni 4,0 filo di sutura tra le due viti di posizionamento della scheda procedura. </li><li> Generare uno spirometro improvvisato rimuovendo e scartando il pistone da tre 1 ml siringhe, e depositando 60 ml di PBS nella parte superiore di ciascun cilindro per formare una chiusura ermetica. Fissare il mozzo del catetere vagamente ad una delle siringhe e posizionarlo su un lato della tavola. </li><li> Aspirare 300 ml di aria in una siringa da 1 ml e posizionarlo su un lato della tavola. </li><li> Tagliare un pezzo aggiuntivo di nastro di circa 6 pollici di lunghezza e metterlo da parte. Questo verrà utilizzato per fissare l&#39;animale alla scheda in fase 2.4. </li><li> Impostare una camera isoflurano. Attaccare O 2, isoflurano, e vuoto per porte appropriate sia sulla camera di esposizione ed il vuoto gioco. In alternativa, somministrare anestetico in un isoflurano compatibile biologicocappa di sicurezza. </li></ol> 2. intubazione <ol><li> Anestetizzare il mouse con isoflurano nella camera fino a quando non perde conoscenza e la respirazione rallenta a un tasso adeguato. Un&#39;esposizione tipico include 4% isoflurano e 2% O 2 da 3 a 4 min, e il risultato ideale è 2 a 2,5 min di sedazione. Ciò corrisponde ad un tasso di respirazione di 1 respiro ogni 2 secondi. </li><li> In attesa che la sedazione per impostare in, aspirare tra 30 e 45 ml di BLM in una pipetta e metterlo da parte. </li><li> Quando si è pronti, sospendere il mouse sedato dai suoi incisivi superiori dal filo attaccato alle viti di posizionamento della piattaforma procedura. Assicurarsi che dorso dell&#39;animale in posizione piana rispetto alla superficie della piattaforma. </li><li> Facendo attenzione a non limitare la ventilazione, posizionare un pezzo di nastro liberamente attraverso il (caudale) parte inferiore della cavità toracica, appena sopra il diaframma. Il posizionamento deve essere abbastanza stretto per mantenere il corretto allineamento durante il procedure, ma non così stretta che limita la respirazione. </li><li> Accendere l&#39;illuminatore tra l&#39;80% e il 100% di intensità e orientare il collo d&#39;oca in modo che sia da 1 a 2 cm dalla superficie della pelle, vicino al plesso solare. Controllare periodicamente la punta di un collo d&#39;oca per il calore per garantire che essa non danneggia il mouse. </li><li> In piedi dietro la piattaforma, utilizzare le sterili, pinze estremità smussata per individuare la lingua. Facendo attenzione ad evitare gli incisivi inferiori, delicatamente presa e disegnare la lingua fuori della cavità orale. </li><li> Utilizzando la mano rimanente, inserire il divaricatore e utilizzarlo per appiattire la lingua contro il pavimento della cavità orale. Rilasciare le pinze, ma lasciare il divaricatore sul posto per i due passaggi successivi. </li><li> Orientare la luce in modo che la trachea è visibile guidando il collo d&#39;oca prossimalmente dal livello del plesso solare fino a raggiungere il livello dei bronchi mainstem. Nota: La trachea può essere facilmente distinta dall&#39;azione della respirazione, WHIch provoca la luce emessa fluttuare in intensità. Quando posizionato correttamente, questa struttura sarà distinguibile nel piano assiale di un perno centrale di luce con luce ambientale minimo nella cavità orale stessa. </li><li> Angle la siringa in modo che segua il percorso naturale della trachea, e abbassare la punta del catetere 22-G, con la siringa attaccata contenente la gocciolina, direttamente nel lume. La bolla PBS inizierà a salire e scendere ad ogni respiro sul posizionamento di successo. Nota: Questa azione può essere ritardata di alcuni secondi a causa della sedazione profonda. </li><li> Nutrire il catetere in un ulteriore 5 mm. Rimuovere la abbassalingua. </li><li> Spostare la siringa per la mano opposta, e impugnando il mozzo, rimuovere delicatamente la siringa. </li><li> Deposito tra 30 e 45 ml di BLM nel centro dell&#39;interno del mozzo del catetere, collegare la seconda siringa ed erogare 300 microlitri di aria nel mozzo. </li><li> Sostituire il secondosiringa con il primo contenente la bolla di PBS. La bolla continuerà a salire e scendere se la procedura è stata eseguita con successo. </li></ol> Cura 3. post-procedurale <ol><li> Rimuovere il catetere e il nastro, e mettere l&#39;animale in un luogo caldo e asciutto fino a quando non riprende conoscenza - di solito entro un paio di minuti. </li></ol>

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A., Aidinis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+pulmonary+fibrosis+with+bleomycin.">Modeling pulmonary fibrosis with bleomycin.</a> Curr Opin Pulm Med. 17, 355-361 (2011).</li><li>Moeller, A., Ask, K., Warburton, D., Gauldie, J., Kolb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bleomycin+animal+model:+a+useful+tool+to+investigate+treatment+options+for+idiopathic+pulmonary+fibrosis?.">The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis?.</a> Int J Biochem Cell Biol. 40, 362-382 (2008).</li><li>Myll&#228;rniemi, M., Kaarteenaho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+treatment+of+idiopathic+pulmonary+fibrosis+-+preclinical+and+clinical+studies+of+pirfenidone,+nintedanib,+and+N-acetylcysteine.">Pharmacological treatment of idiopathic pulmonary fibrosis - preclinical and clinical studies of pirfenidone, nintedanib, and N-acetylcysteine.</a> European Clinical Respiratory Journal. 2, (2015).</li><li>Reinert, T., Baldotto, C. S. d. R., Nunes, F. A. P., Scheliga, A. A. d. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleomycin-Induced+Lung+Injury.">Bleomycin-Induced Lung Injury.</a> Journal of Cancer Research. 2013, 1-9 (2013).</li><li>Lakatos, H. 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=Oropharyngeal+aspiration+of+a+silica+suspension+produces+a+superior+model+of+silicosis+in+the+mouse+when+compared+to+intratracheal+instillation.">Oropharyngeal aspiration of a silica suspension produces a superior model of silicosis in the mouse when compared to intratracheal instillation.</a> Exp Lung Res. 32, 181-199 (2006).</li><li>Helms, M. N., Torres-Gonzalez, E., Goodson, P., Rojas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+tracheal+instillation+of+solutes+into+mouse+lung.">Direct tracheal instillation of solutes into mouse lung.</a> J Vis Exp. , (2010).</li><li>Osier, M., Oberdorster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+inhalation+vs+intratracheal+instillation:+differences+in+particle+effects.">Intratracheal inhalation vs intratracheal instillation: differences in particle effects.</a> Fundam Appl Toxicol. 40, 220-227 (1997).</li><li>Driscoll, K. E., 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=Intratracheal+instillation+as+an+exposure+technique+for+the+evaluation+of+respiratory+tract+toxicity:+Uses+and+Limitations.">Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: Uses and Limitations.</a> Toxicol Sci. 55, 24-35 (2000).</li><li>MacDonald, K. D., Chang, H. Y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+simple+method+of+mouse+lung+intubation.">An improved simple method of mouse lung intubation.</a> J Appl Physiol (1985). 106, 984-987 (1985).</li><li>Spoelstra, E. N., 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+novel+and+simple+method+for+endotracheal+intubation+of+mice.">A novel and simple method for endotracheal intubation of mice.</a> Lab Anim. 41, 128-135 (2007).</li><li>Cai, Y., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+intratracheal+intubation+to+study+the+pathology+and+physiology+of+mouse+lung.">Noninvasive intratracheal intubation to study the pathology and physiology of mouse lung.</a> J Vis Exp. , e50601 (2013).</li><li>Starcher, B., WIlliams, I. <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+for+intratracheal+instillation+of+endotoxin+into+the+lungs+of+mice.">A method for intratracheal instillation of endotoxin into the lungs of mice.</a> Lab Anim. 23, 234-240 (1989).</li><li>Daubeuf, F., Frossard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+bronchoalveolar+lavage+in+the+mouse.">Performing bronchoalveolar lavage in the mouse.</a> Curr Protoc Mouse Biol. 2, 167-175 (2012).</li><li>Li, 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=Severe+lung+fibrosis+requires+an+invasive+fibroblast+phenotype+regulated+by+hyaluronan+and+CD44.">Severe lung fibrosis requires an invasive fibroblast phenotype regulated by hyaluronan and CD44.</a> J Exp Med. 208, 1459-1471 (2011).</li><li>Grazioli, S., 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=CYR61+(CCN1)+overexpression+induces+lung+injury+in+mice.">CYR61 (CCN1) overexpression induces lung injury in mice.</a> Am J Physiol Lung Cell Mol Physiol. 308, L759-L765 (2015).</li><li>Redente, E. 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The authors have nothing to disclose.

Nei casi in cui aerosol è impraticabile a causa della limitata disponibilità di reagente, la sicurezza, o il costo, amministrazione diretta tracheale è un metodo superiore per la consegna degli agenti esogeni nei polmoni 16 transtracheale instillazione è stato ampiamente utilizzato per raggiungere questo obiettivo.; Tuttavia, come con tutti gli interventi chirurgici, porta con sé anche il potenziale di complicazioni causate dalla procedura stessa, e non necessariamente l&#39;agente essendo instillato. <s...

Nonostante alcune differenze anatomiche e fisiologiche, 1 modelli murini continuano ad essere prezioso per la modellazione di biologia umana e patogenesi della malattia. 2 Dal punto di vista allevamento, i topi sono facili da gestire, hanno un basso tempo di allevamento, una durata di vita accelerato, e sono relativamente poco costoso a casa. Con lo sviluppo di diversi ceppi genetici e strategie (ad es., Condizionali knock-out, topi reporter, approcci lineage tracing, ecc), così come l&#39;ampia gamma di reagenti disponibili (ad es., Anticorpi, proteine ​​ricombinanti, inibitori, ecc), i topi sono diventati un elemento essenziale organismo modello vertebrato per scoprire i processi di omeostasi e malattie umane. 3 I topi sono stati particolarmente preziosi per lo studio le malattie polmonari, tra cui lesione acuta polmonare (ALI) e fibrosi polmonare. 4 ALI negli esseri umani può essere causata da traumi, lesioni, o sepsi ed è caratterizzata da epiteliale eperdita endoteliale (es., edema), l&#39;infiammazione e la fibrosi nascente. In molti pazienti, ALI progredisce nella sua forma grave, sindrome da distress respiratorio acuto (ARDS), che si traduce spesso in fibrosi e la morte a causa di insufficienza respiratoria. 5,6 fibrosi polmonare è una progressiva, una patologia fatale caratterizzata dall&#39;eccesso deposizione di matrice extracellulare , in particolare collagene di tipo I, portando a deficit della funzionalità respiratoria. 7,8 somministrazione di bleomicina (BLM) è il modello più diffuso e meglio caratterizzato per indurre ALI e fibrosi negli animali da esperimento. 9 Sebbene BLM-indotta fibrosi polmonare nei roditori fa non ricapitolare completamente i fenotipi umani fibrotiche, 10 studi di topo con questo modello hanno portato alla scoperta di importanti fattori che influenzano l&#39;insorgenza e la progressione della malattia. 11 Mentre l&#39;esatto meccanismo (s) dietro fibrogenesi BLM-indotta sono sconosciuti, il danno avvioè pensato per sorgere dal contatto-dipendente rotture del DNA nelle cellule epiteliali che rivestono le vie aeree di conduzione e alveoli, in particolare, di tipo 1 pneumociti. 12 Il bisogno di contatto diretto tra BLM e l&#39;epitelio polmonare mette in evidenza l&#39;importanza di un robusto percorso di consegna , e queste preoccupazioni sono anche germano a una vasta gamma di trattamenti mirati per le vie aeree distali, tra cui proteine ​​ricombinanti, anticorpi, siRNA, virus, batteri, particelle, e altro ancora. Aspirazione orofaringeo (OPA) è stato ampiamente utilizzato per questo scopo 13, ma un importante una lacuna di OPA è che una parte dell&#39;agente erogata sia assorbito nel tratto gastrointestinale, determinando così imprecisione della dose somministrata. Un altro approccio ampiamente utilizzato è instillazione transtracheale, che coinvolge tracheostomia sotto forte anestesia per esporre la trachea e instillazione di un agente direttamente nelle vie respiratorie. 14 Tuttavia, non solo può taleuna procedura sia indesiderabile a causa della sua invasività, ma è anche in termini di tempo, richiede un bel po &#39;di allenamento, e provoca una lesione potente per le vie respiratorie sono stati sviluppati. 15,16 Diversi protocolli che coinvolgono l&#39;amministrazione diretta di agenti in trachea senza la necessità di un intervento chirurgico, 16,17,18,19,20 tempi di recupero ma questi metodi comportano estesi causato da anestetici potenti, l&#39;uso di attrezzature costose (es., otoscope / laringoscopio, disponibili in commercio tavole procedura, fibra ottica fili, ecc), un eccesso di manipolazione nella cavità orale, l&#39;incertezza del dosaggio. Questo documento descrive un metodo relativamente semplice di somministrazione tramite intubazione che permette un ricercatore modo rapido, economico e affidabile infondere un reagente nel polmone murino con limitato rischio di danno residuo ai tessuti circostanti.

<table><tbody><tr><td>Bleomycin For Injection, 30 units/vial</td><td>APP Pharmaceuticals, LLC</td><td>103720</td><td>For best results, BLM should be suspended in PBS, aliquoted, and stored as single use lyophilzed aliquots</td></tr><tr><td>Blunt End Forceps</td><td></td><td></td><td></td></tr><tr><td>Tongue Depressor (i.e. bent Valleylab Blade Electrode, 2.4&quot;)&amp;nbsp;</td><td>Covidien</td><td>E1551G</td><td>Before use, create a 45 degree bend 1.5 cm&amp;nbsp; from the blade tip. A suitable depressor can also be created from any metal implement of similar dimensions.&amp;nbsp;</td></tr><tr><td>Exel Safelet Catheter 22G X 1&quot;</td><td>Exel International</td><td>26746</td><td></td></tr><tr><td>1 ml Slip-tip Disposable Tuberculin Syringe (200/sp, 1600/ca)</td><td>BD</td><td>309659</td><td></td></tr><tr><td>0.2 ml Pipettor and Filter Tips</td><td></td><td></td><td></td></tr><tr><td>Fiber-Lite Illuminator. Model 181-1: Model 180 mated with Standard Dual Gooseneck illuminator</td><td>Dolan Jenner Industries, Inc.</td><td>181-1</td><td>Lower output LED illuminators are not recommended as they fail to suficiently illuminate the trachea.</td></tr><tr><td>Intubation Board</td><td></td><td></td><td>See Diagram 1.</td></tr><tr><td>Colored Label Tape: 0.5&amp;nbsp;in. Wide</td><td>Fisherbrand</td><td>15-901-15A</td><td></td></tr><tr><td>Oxygen&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Phosphate-Buffered Saline, 1X</td><td>Corning</td><td>21-040-CV</td><td>Product should be sterile</td></tr><tr><td>Non-Sterile Silk Black Braided Suture Spool, 91.4 m, Size 4-0</td><td>Harvard Apparatus</td><td>517698</td><td></td></tr><tr><td>Table Top Anesthesia Machine Isoflurane</td><td>Highland Medical Equipment</td><td></td><td>http://www.highlandmedical.net/</td></tr><tr><td>Slide Top Induction Mouse Isoflurane Chamber</td><td>MIP / Anesthesia Technologies</td><td>AS-01-0530-SM</td><td></td></tr><tr><td>FORANE (isoflurane, USP) Liquid For Inhalation 100 mL&amp;nbsp;</td><td>Baxter</td><td>1001936040</td><td></td></tr><tr><td>&amp;nbsp;Nanozoomer Digital Pathology system</td><td>Hamamatsu</td><td></td><td></td></tr><tr><td>IgM ELISA Quantification Kit&amp;nbsp;</td><td>Bethyl Laboratories</td><td>E90-101</td><td></td></tr></tbody></table>

an improved method for rapid intubation of the trachea in mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

Topi intubati sono stati monitorati giornalmente per la perdita di peso e di angoscia, e si sono sacrificati 4, 10 o 17 giorni dopo tramite iniezione intraperitoneale di 2,5% 2,2,2- tribromoethanol. Il lavaggio broncoalveolare (BAL) è stato raccolto in tre lavaggi di PBS come descritto altrove 21 , e il polmone destro è stato fissato in formalina 10%, inclusi in paraffina, e colorati con tricromica di Masson dalla University of Washington Istologia ed Imagi...

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An Improved Method for Rapid Intubation of the Trachea in Mice

Un metodo migliore per Rapid intubazione della trachea nei topi

Questo articolo presenta un metodo rapido e semplice per l&#39;amministrazione bleomicina direttamente nella trachea del mouse tramite intubazione. I principali vantaggi di questo metodo sono che è altamente riproducibile, facile da padroneggiare, e non richiede attrezzature specializzate o tempi di recupero lunghi.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

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JoVE Journal

Medicine

25.1K Views.  University of Washington.  Questo articolo presenta un metodo rapido e semplice per l'amministrazione bleomicina direttamente nella trachea del mouse tramite intubazione. I principali vantaggi di questo metodo sono che è altamente riproducibile, facile da padroneggiare, e non richiede attrezzature specializzate o tempi di recupero lunghi. 

Video: An Improved Method for Rapid Intubation of the Trachea in Mice

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Pressione di ventilazione controllata per indurre lesioni polmonari acute nei topi

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Ricerca

Medicina

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G&#160;intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.

Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope

We have developed a simple, reliable, and relatively inexpensive method for endotracheal intubation in mice via direct laryngoscopy using an otoscope with a 2.0 mm speculum. This technique is atraumatic and can be used for repeated measurements in chronic experiments. We find it superior to tracheostomy or previously reported nonsurgical techniques.

Intubazione endotracheale in Mouse<em&gt; Via</em&gt; Laringoscopia diretta usando un otoscopio

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

A Simple Method of Mouse Lung Intubation

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

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

Un metodo semplice di intubazione mouse polmone

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

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.

Non invasivo intratracheale intubazione per studiare la Patologia e Fisiologia del mouse polmone

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 ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

Un reversibile, metodo non invasivo per le misure di resistenza delle vie aeree e liquido di lavaggio broncoalveolare di campionamento nei topi

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Biologia

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Un modello chiuso-cassa per indurre costrizione trasversale aortica in topi

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

Un modello murino di intubazione orotracheale e chirurgia ventilata di ischemia polmonare riperfusione

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

Immunologia e infezioni

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imaging del calcio nell'oliva inferiore del topo

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Neuroscienze

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

Intubazione orotrachea ripetuta nei topi

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

<meta charset="utf8"></head><body>Protocollo di intubazione endotracheale basato sulla tracheotomia per modelli di ratto non sopravviventi

Endotracheal Intubation via Tracheotomy and Subsequent Thoracotomy in Rats for Non-Survival Applications

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that require controlled or high ventilation pressures or access to the thoracic cavity and organs. Conventional endoorotracheal intubation using the anatomically existing route through the mouth is well suited for survival experiments. However, this procedure poses some challenges, including generally higher levels of the required experience and technical skill, more advanced equipment, and greater time effort with relevant intubation failure rates and complications such as tracheal perforation, temporary systemic hypooxygenation, and relevant aerial leakage.
This manuscript, therefore, presents a detailed step-by-step protocol for endotracheal intubation through tracheotomy in non-survival rat models when guaranteed intubation success, constant oxygenation levels, high ventilation pressures, or open thoracotomy are required.
The protocol emphasizes the importance of meticulous surgical technique to ensure consistent and reliable outcomes, especially for researchers who are inexperienced or lack routine in the technique of endoorotracheal intubation via direct laryngoscopy. This procedure is, therefore, expected to minimize animal suffering and unnecessary animal losses.

<meta charset="utf8"></head><body>Intubazione endotracheale tramite tracheotomia e successiva toracotomia nei ratti per applicazioni non di sopravvivenza

Here we introduce a standardized procedure for endotracheal intubation via tracheotomy followed by thoracotomy in rats, aimed at enhancing the precision and reproducibility of non-survival applications requiring invasive ventilation and exposure of thoracic organs in in vivo rat models.

Intubazione endotracheale tramite tracheotomia e successiva toracotomia nei ratti per applicazioni non di sopravvivenza

Endotracheal intubation and subsequent ventilation are often basic requirements for translational research in rat models for various interventions that ...