Name: a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation
Uploaded: 2017-07-26T15:00:00.000+00:00
Duration: 8 min 46 s
Description: Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

Gli autori vorrebbero riconoscere all&#39;Istituto Nazionale di Scienze Biologiche di Pechino per l&#39;assistenza. Questo lavoro è stato sostenuto da Beijing Municipal Natural Science Foundation (numero Z17110200040000).

Tutte le procedure utilizzate in questo protocollo sono state eseguite in conformità alle raccomandazioni contenute nelle Linee Guida per la cura e l&#39;uso degli animali da laboratorio dell&#39;Istituto Nazionale di Scienze Biologiche di Pechino. I topi maschi CD-1 di 8 settimane sono stati alloggiati in una struttura specifica senza patogeni (SPF) fino all&#39;esecuzione degli esperimenti. Le chirurgie sono state eseguite utilizzando topi completamente anestetizzati ( cioè , senza risposte a pizzicotto). Dopo l&#39;intervento chirurgico, i topi sono stati tenuti in una stanza calda e umida con cibo sufficiente e acqua dolce. I topi sono stati sacrificati usando un sovradosaggio di anestetico erogato mediante iniezione intraperitoneale. 1. Mouse PNX Chirurgia <ol><li> Completa l&#39;anestesia dei topi con fenobarbital di sodio (120 mg / kg di peso corporeo) e buprenorfina (0,1 mg / kg di peso corporeo) tramite l&#39;iniezione intraperitoneale (IP). Effettuare la chirurgia quando i topi non reagiscono più a pizzicamento del piede. </li><li> Togliere i capelli sul torace sinistro dei topi con la sostanza chimicaTrattamento infettivo (~ 3 x 3 cm 2 area). </li><li> Fissare ogni mouse su una piattaforma di intubazione con il lato ventrale rivolto verso l&#39;operatore ( Figura 1A ). </li><li> Estrarre la linguetta del mouse e illuminare le corde vocali con un piccolo laringoscopio animale contenente una tacca per guidare i cateteri 18 ( Figura 1A ). </li><li> Distinguete le corde vocali osservando i movimenti delle corde vocali durante la respirazione. Inserire delicatamente una cannula intubazione endovenosa da 20 G nella trachea ad un angolo anteriore 19 . </li><li> Posizionare i topi in una posizione reclinabile laterale destra e collegare la cannula ad un ventilatore meccanico ( ad esempio, a pressione controllata, vedere la tabella dei materiali ). Controllare l&#39;inserimento della cannula nella trachea osservando i movimenti respiratori del petto del mouse ( Figura 1B ). </li><li> Impostare la pressione inspiratoria oF il ventilatore a 12 cm H 2 O e impostare la frequenza respiratoria a 120 breath per min ( Figura 1B ). </li><li> Decontaminare la pelle nell&#39;area chirurgica con betadina e il 70% di etanolo. </li><li> Effettuare un&#39;incisione toracotomica posterolaterale da 2 - 3 cm nello spazio al 5 ° spazio intercostale, tagliato attraverso la pelle e i muscoli con forbici a molla Noyes (tagliente: 14 mm; diametro punta: 0,275 mm) ( Figura 2B , C ). Gli strumenti chirurgici utilizzati per la procedura toracotomia vengono sterilizzati prima dell&#39;uso. </li><li> Effettuare un&#39;incisione di 1,5 cm al 5 ° spazio intercostale per esporre il polmone sinistro ( Figura 2D , E ). Durante l&#39;operazione, utilizzare un cauterizzatore ad alta temperatura per bloccare l&#39;emorragia. </li><li> Sollevare un terzo del lobo polmonare sinistro dal torace con punte punte sbilanciate ( Figura 2F ), quindi utilizzare un tampone di cotone per estrarre l&#39;intera sinistraPolmone ( Figura 2G ). </li><li> Identificare l&#39;arteria polmonare ei bronchi del lobo polmonare sinistro ( Figura 2G ). </li><li> Legare leggermente i bronchi ei vasi all&#39;hilum con una sutura chirurgica di seta e tagliare il lobo polmonare sinistro a 3 - 4 mm dalla legatura ( Figura 2H , I ). NOTA: Fare attenzione a non tagliare i nodi di sutura sull&#39;hilum sinistro, che possono causare pneumotorace ( ossia aria o gas nella cavità del torace) . </li><li> Chiudere la parete toracica con 1 sutura e quindi cucire lo strato muscolare e lo strato della pelle in sequenza, utilizzando 5 - 6 suture interrotte. Lasciare uno spazio tra 3 e 4 mm tra ciascuna sutura ( figura 2M , 2N ). NOTA: Tenere l&#39;ago chirurgico di sutura lontano dal cuore; La puntura cardiaca involontaria provoca la morte immediata. </li><li> Disinfettare l&#39;area chirurgica con povidone-iodio. </li><li> afteR l&#39;operazione chirurgica, posizionare il mouse su un tampone termico a 38 ° C e collegare il mouse al ventilatore fino a quando non inizieranno i movimenti respiratori spontanei ( Figura 2O ). </li></ol> 2. Implantologia della protesi <ol><li> Eseguire i passaggi 1.1-1.13 della procedura PNX (cioè fino al punto in cui il lobo polmonare sinistro del mouse viene rimosso). </li><li> Fissare il centro della protesi in silicone (cliente realizzato, 12 mm di lunghezza, 3 mm di spessore, 7 mm di larghezza, 0,2 g, forma ellisse) utilizzando pinze a punta ( Figura 2J ). Sterilizzare la protesi al silicone prima dell&#39;inserimento. </li><li> Tenere la nervatura con le pinze con una mano per esporre la cavità toracica e poi inserire la protesi nella cavità toracica vuota sinistra con un&#39;altra mano. NOTA: L&#39;angolo di inserzione è di circa 45 gradi tra il piano frontale della protesi e la superficie toracica ( Figura 2K ,L). Essere molto gentile quando si inserisce la protesi. La forza eccessiva provoca una rottura pleurica. </li><li> Regolare l&#39;orientamento della protesi con pinze sbilanciate per assicurare che la protesi occupa la cavità toracica vuota sinistra. </li><li> Eseguire i passaggi 1.14 - 1.16 della procedura PNX del mouse. </li></ol> 3. Misurazione dell&#39;ILV <ol><li> Preparare un dispositivo personalizzato (&quot;tubo di inflazione&quot;) costituito da uno stantuffo rimosso da una pipetta serologica usa e getta (10 mL), un tubo flessibile lungo 40 cm con adattatore a ago, valvola di controllo della portata e un ago da 18 G. Dopo l&#39;assemblaggio, fissare la pipetta su una scheda con nastro ( Figura 3A ). La distanza tra la parte superiore della pipetta e la panca sperimentale deve essere di almeno 30 cm. </li><li> Preparare la soluzione di fissazione del 4% di paraformaldeide (PFA), sciogliendo 20 g di PFA in 500 ml di soluzione salina fosforata tamponata pre-riscaldata (PBS) in un bagno d&#39;acqua di 55 ° C, scuotendo manualmenteCe ogni 10 minuti fino a quando la soluzione è chiara. Dopo raffreddamento a temperatura ambiente, filtrare la soluzione con un filtro da 0,45 μm. ATTENZIONE: Usare un adeguato dispositivo di protezione individuale (PPE) quando si maneggia PFA. </li><li> Mouse sacrificato con un&#39;iniezione di sovradosaggio di anestetico (0,8% fenobarbital sodico, 1000 U / ml di eparina). </li><li> Fissare ogni mouse su una piastra di dissezione in polistirene e spruzzarla con alcool al 70%. </li><li> Aprire con cautela il petto del mouse e tagliare lo sterno con forbici per esporre completamente i lobi del polmone. </li><li> Rimuovere il tessuto eccessivo con forbici per esporre la trachea. Assicuratevi di separare la trachea dall&#39;esofago. </li><li> Tagliare l&#39;aorta addominale e inserire un ago da 25 gauge nel ventricolo destro del cuore; Collegare l&#39;ago ad una siringa da 20 ml prima di questo inserimento. Spingere lentamente 1x PBS nel cuore per rimuovere le cellule del sangue fino a quando i polmoni diventano bianchi. Tipicamente, 5 - 10 mL di PBS è necessario per eliminare i vasi sanguigni polmonari. </li><li> Compilare il thE tubo di infusione personalizzato con 4% di PFA fresco e rimuovere tutte le bolle dal tubo di inflazione. </li><li> Inserire l&#39;ago da 18 gauge del tubo di infusione nella trachea e agganciare la trachea con le clip del recipiente per evitare perdite di liquido. </li><li> Gonfiare i polmoni con il 4% di PFA ad una pressione costante di 25 cm / H 2 O 2 , 20 . Incubare i polmoni a temperatura ambiente per 2 ore per ottenere polmoni completamente espansi. Questo passo &quot;pre-fix&quot; è fondamentale per la conservazione della morfologia polmonare. <ol><li> Monitorando il tubo di inflazione, registrare il valore del volume iniziale del 4% PFA e registrare il volume finale. Il volume polmonare interno equivale al volume iniziale del PFA del 4% meno il volume PFA finale del 4%. </li></ol></li><li> Legare la trachea e usando le forbici, disseccare delicatamente i polmoni (mantenendo intatti i polmoni) dai tessuti connettivi circostanti. Essere molto gentile per evitare di danneggiare i polmoni. </li><li> incuVersare i polmoni in un tubo conico da 50 ml riempito con 4% di PFA per 12 h a 4 ° C con una leggera agitazione su un agitatore (50 rpm). Procedere alla lavorazione del tessuto e alla colorazione (vedere la sezione 4). </li></ol> 4. Incorporazione del tessuto, sezionamento e colorazione di ematossilina &amp; eosina (H &amp; E) <ol><li> Dopo la fissazione, utilizzare le forbici forensi di Noyes per tagliare il cuore e i tessuti connettivi eccessivi dai polmoni. Separare delicatamente i singoli lobi del polmone tagliando il bronco che collega i lobi del polmone alla trachea. </li><li> Lavare ampiamente i lobi del polmone 3 - 4 volte in 50 ml di 1x PBS (30 minuti / lavaggio) su un agitatore orbitale (50 rpm). </li><li> Dopo il lavaggio finale, crioprire i lobi del polmone immergendoli in una soluzione al 30% di saccarosio (in 1x PBS) a 4 ° C fino a quando il tessuto si affonda sul fondo dei tubi conici da 50 ml (circa 12 h). </li><li> Prima di incollare e criocondire i tessuti, rimuovere i campioni del lobo polmone dai tubi con le pinze, mantenere l&#39;accesI lobi per l&#39;analisi istologica, immergere la residua soluzione di saccarosio dalla superficie dei campioni del lobo accessorio e quindi immergere completamente il campione in un piatto di Petri contenente il composto ottimale di taglio (OCT) per circa 30 minuti. </li><li> Bloccare i campioni di lobi accessorio incorporati in OCT in azoto liquido usando criomaglie. Posizionare la superficie più grande del lobo parallela alla parte inferiore dello stampo. </li><li> Preparare un totale di tre sezioni di 10 μm di spessore per ciascun campione durante la crizione per l&#39;analisi istologica. Scartare il primo 1 mm di tessuto, raccogliere una sezione di 10 μm di spessore, scartare 0,5 mm di tessuto, raccogliere un&#39;altra sezione, scartare 0,5 mm di tessuto e raccogliere la terza sezione (finale). </li><li> Aria asciugare le sezioni per 1 ora prima di eseguire la colorazione H &amp; E. </li><li> Effettuare la colorazione H &amp; E <ol><li> Lavare le sezioni in 3 - 4 cambi di acqua di rubinetto e poi macchia le sezioni in fresco ematoxilina per 2 min; Sciacquare la sezione sotto l&#39;acqua corrente del rubinetto; Immergere la sezione due volte in una soluzione 1% HCl-70% di etanolo per rimuovere l&#39;ematoxilina in eccesso. </li><li> Macchiare la sezione in eosin fresco per 3 min; Deidratare le sezioni con due successive lavaggi a 30 s in etanolo al 95% e due lavaggi a 30 s con etanolo al 100%; Chiudere le sezioni in xilene per 30 s, ripetere la fase di compensazione una volta in xilene fresco; Montare le diapositive con il supporto di montaggio usando le copertine di vetro. </li></ol></li></ol> 5. Quantificazione di MLI <ol><li> Acquisire immagini digitali delle sezioni Lobe accessorio macchiate H &amp; E (ingrandimento 20X) utilizzando un microscopio a campo luminoso. </li><li> Per quantificare il MLI, selezionare un totale di 15 viste non sovrapposte (1.000 μm x 1.000 μm) in modo casuale dalle aree adattate (senza arterie e vene, vie respiratorie principali e condotti alveolari) di 3 sezioni. </li><li> Inserire una griglia con 10 linee verticali distribuite uniformemente e 10 linee orizzontali distribuite equamente distribuite di lenGth (1.000 μm) sulle aree di vista prescelte usando uno strumento di righello; Ogni linea è così distanziata da 100 μm di distanza ( Figura 4B ). </li><li> Definire il valore di una intercetta come la lunghezza lineare tra due epiteli alveolari adiacenti. Misurare i valori di tutte le intercettazioni lungo ciascuna linea di lunghezza di 1000 μm. </li><li> Per ciascuna griglia, quantificare i valori di tutte le intercetta tra le 10 lunghezze orizzontali di lunghezza 1.000 μm e le 10 lunghezze verticali di 1.000 μm. NOTA: MLI è il valore medio delle lunghezze di intercettazione da un totale di 15 griglie analizzate tra le tre sezioni predisposte per ciascuno dei lobi accessori. </li></ol> 6. Calcolo dell&#39;ISA <ol><li> Calcola l&#39;ISA utilizzando l&#39; equazione 1 (vedi Introduzione ). Fare riferimento alla sezione 3 per la misura di ILV e fare riferimento alla sezione 5 per la quantificazione di MLI. </li></ol>

<ol>
	<li>Liu, Z., 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=MAPK-Mediated+YAP+Activation+Controls+Mechanical-Tension-Induced+Pulmonary+Alveolar+Regeneration.">MAPK-Mediated YAP Activation Controls Mechanical-Tension-Induced Pulmonary Alveolar Regeneration.</a> Cell Rep. 16 (7), 1810-1819 (2016).</li><li>Thurlbeck, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+surface+area+and+other+measurements+in+emphysema.">Internal surface area and other measurements in emphysema.</a> Thorax. 22 (6), 483-496 (1967).</li><li>Knudsen, L., Weibel, E. R., Gundersen, H. J. G., Weinstein, F. V., Ochs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+air+space+size+characteristics+by+intercept+(chord)+measurement:+an+accurate+and+efficient+stereological+approach.">Assessment of air space size characteristics by intercept (chord) measurement: an accurate and efficient stereological approach.</a> J Appl Physiol. 108 (2), 412-421 (2010).</li><li>Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Morphometry of the Human Lung. , (1963).</li><li>Duguid, J. B., Young, A., Cauna, D., Lambert, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+internal+surface+area+of+the+lung+in+emphysema.">The internal surface area of the lung in emphysema.</a> J Pathol Bacteriol. 88, 405-421 (1964).</li><li>Branchfield, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+neuroendocrine+cells+function+as+airway+sensors+to+control+lung+immune+response.">Pulmonary neuroendocrine cells function as airway sensors to control lung immune response.</a> Science. 351 (6274), 707-710 (2016).</li><li>Ding, B. -. 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=Endothelial-derived+angiocrine+signals+induce+and+sustain+regenerative+lung+alveolarization.">Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization.</a> Cell. 147 (3), 539-553 (2011).</li><li>Dunnill, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+methods+in+the+study+of+pulmonary+pathology.">Quantitative methods in the study of pulmonary pathology.</a> Thorax. 17 (4), 320-328 (1962).</li><li>Weibel, E. R., Gomez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+human+lung.+Use+of+quantitative+methods+establishes+fundamental+relations+between+size+and+number+of+lung+structures.">Architecture of the human lung. Use of quantitative methods establishes fundamental relations between size and number of lung structures.</a> Science. 137 (3530), 577-585 (1962).</li><li>Thurlbeck, W. 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+internal+surface+area+of+nonemphysematous+lungs.">The internal surface area of nonemphysematous lungs.</a> Am Rev Respir Dis. 95 (5), 765-773 (1967).</li><li>Butler, J. 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=Evidence+for+adult+lung+growth+in+humans.">Evidence for adult lung growth in humans.</a> N Engl J Med. 367 (16), 244-247 (2012).</li><li>Hsia, C. C. W., Herazo, L. F., Fryder-Doffey, F., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compensatory+lung+growth+occurs+in+adult+dogs+after+right+pneumonectomy.">Compensatory lung growth occurs in adult dogs after right pneumonectomy.</a> J Clin Invest. 94 (1), 405-412 (1994).</li><li>Thurlbeck, S. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumonectomy+in+Rats+at+Various+Ages.">Pneumonectomy in Rats at Various Ages.</a> Am Rev Respir Dis. 120 (5), 1125-1136 (1979).</li><li>Cagle, P. T., Langston, C., Thurlbeck, W. 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+Effect+of+Age+on+Postpneumonectomy+Growth+in+Rabbits.">The Effect of Age on Postpneumonectomy Growth in Rabbits.</a> Pediatr Pulmonol. 5 (2), 92-95 (1988).</li><li>Langston, C., 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=Alveolar+multiplication+in+the+contralateral+lung+after+unilateral+pneumonectomy+in+the+rabbit.">Alveolar multiplication in the contralateral lung after unilateral pneumonectomy in the rabbit.</a> Am Rev Respir Dis. 115 (1), 7-13 (1977).</li><li>Cohn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+Affecting+The+Postnatal+Growth+of+The+Lung.">Factors Affecting The Postnatal Growth of The Lung.</a> Anatomical Record. 75 (2), 195-205 (1939).</li><li>Hsia, C. C., Wu, E. Y., Wagner, E., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+mediastinal+shift+after+pneumonectomy+impairs+regenerative+alveolar+tissue+growth.">Preventing mediastinal shift after pneumonectomy impairs regenerative alveolar tissue growth.</a> Am J Physiol Lung Cell Mol Physiol. 281 (5), L1279-L1287 (2001).</li><li>Das, S., MacDonald, K., Chang, H. -. Y. S., 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=A+simple+method+of+mouse+lung+intubation.">A simple method of mouse lung intubation.</a> J Vis Exp. (73), e50318 (2013).</li><li>Liu, S., Cimprich, J., Varisco, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+pneumonectomy+model+of+compensatory+lung+growth.">Mouse pneumonectomy model of compensatory lung growth.</a> J Vis Exp. (94), (2014).</li><li>Silva, M. F. R., Zin, W. A., Saldiva, P. H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airspace+configuration+at+different+transpulmonary+pressures+in+normal+and+paraquat-induced+lung+injury+in+rats.">Airspace configuration at different transpulmonary pressures in normal and paraquat-induced lung injury in rats.</a> Am J Respir Crit Care Med. 158 (4), 1230-1234 (1998).</li><li>Yilmaz, C., 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=Noninvasive+quantification+of+heterogeneous+lung+growth+following+extensive+lung+resection+by+high-resolution+computed+tomography.">Noninvasive quantification of heterogeneous lung growth following extensive lung resection by high-resolution computed tomography.</a> J Appl Physiol. 107 (5), 1569-1578 (2009).</li><li>Voswinckel, R., 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=Characterisation+of+post-pneumonectomy+lung+growth+in+adult+mice.">Characterisation of post-pneumonectomy lung growth in adult mice.</a> Eur Respir J. 24 (4), 524-532 (2004).</li><li>Ravikumar, 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=Regional+Lung+Growth+and+Repair+Regional+lung+growth+following+pneumonectomy+assessed+by+computed+tomography.">Regional Lung Growth and Repair Regional lung growth following pneumonectomy assessed by computed tomography.</a> J Appl Physiol. 97, 1567-1574 (2004).</li><li>Gibney, B. C., 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=Detection+of+murine+post-pneumonectomy+lung+regeneration+by+18FDG+PET+imaging.">Detection of murine post-pneumonectomy lung regeneration by 18FDG PET imaging.</a> EJNMMI Res. 2 (1), (2012).</li><li>Mu&#241;oz-Barrutia, A., Ceresa, M., Artaechevarria, X., Montuenga, L. M., Ortiz-De-Solorzano, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+lung+damage+in+an+elastase-induced+mouse+model+of+emphysema.">Quantification of lung damage in an elastase-induced mouse model of emphysema.</a> Int J Biomed Imaging. 2012, (2012).</li></ol>

Gli autori non hanno niente da rivelare.

In questo protocollo forniamo descrizioni dettagliate sulla misura dei parametri polmonari dopo il polmone a sinistra del polmone PNX e l&#39;impianto di protesi. L&#39;ISA è ora considerata una metrica chiave per la valutazione della funzione respiratoria in molte malattie polmonari e nella rigenerazione alveolare indotta da lesioni. Tuttavia, sebbene la comunità di ricerca polmonare sia d&#39;accordo sull&#39;utilità dell&#39;ISA come metrica utile, fino ad oggi non è stata presa in considerazione la standardizzaz...

La funzione fondamentale del polmone è lo scambio di ossigeno e di anidride carbonica tra i vasi sanguigni e l&#39;atmosfera. Le malattie polmonari come la displasia broncopolmonare (BPD), la malattia polmonare cronica ostruttiva (COPD) e le infezioni respiratorie acute provocano una riduzione dell&#39;ISA 2 . I ricercatori che studiano la malattia polmonare hanno sviluppato diversi metodi quantitativi per valutare i cambiamenti morfologici nei polmoni, tra cui MLI, ILV, numero di scambiatori di gas, ISA e conformità ai tessuti polmonari 2 , 3 . Studi pionieristici di Weibel et al. 4 e Duguid et al. 5 hanno stabilito che l&#39;ISA può essere utilizzata come misura diretta della capacità di scambio del gas polmonare nei polmoni umani e può essere utilizzata come criterio per determinare la gravità dell&#39;emfisema. Alcuni studi pubblicati negli ultimi cinque anni hanno utilizzato parametri morfologici polmonari ( ad esempio, </eM&gt; ISA e MLI) per valutare i cambiamenti morfologici e funzionali nei polmoni dei topi durante lo sviluppo 6 e durante il recupero da lesioni PNX 1 , 7 . L&#39;ISA viene calcolato utilizzando l&#39; equazione 1 8 , 9 : <img alt="Equazione" src="/files/ftp_upload/56114/56114eq1.jpg" /> , Dove ILV è il volume polmonare interno e MLI è un parametro intermedio che rappresenta la dimensione aerospaziale periferica polmonare 10 . La PNX, la rimozione chirurgica di uno o più lobi del polmone, è stata ampiamente riportata per indurre la rigenerazione alveolare in molte specie, tra cui gli esseri umani 11 , i topi 1 , i cani 12 , i ratti 13 e i conigli 14 e 15 . Un studY dei polmoni dei topi a quattordici giorni dopo il PNX ha mostrato che sia l&#39;espansione di alveoli preesistenti e la de novo formazione di alveoli contribuiscono al restauro di ISA, ILV e il numero di alveoli nei restanti tessuti polmonari 1 . Noi e altri abbiamo dimostrato che l&#39;inserimento di materiali come la spugna, la cera o una protesi a forma personalizzata nella cavità toracica vuota dopo PNX ( cioè l&#39; impianto di protesi) compromette la rigenerazione alveolare. È ora stabilito che la forza meccanica funziona come uno dei fattori più importanti per iniziare la rigenerazione alveolare 1 , 16 , 17 . Tali studi hanno evidenziato l&#39;efficacia dell&#39;uso dei valori ISA da polmoni trattati con PNX e protesi da protesi come criterio per valutare quantitativamente la rigenerazione alveolare. Il bias dell&#39;osservatore è noto per influenzare in modo significativo la misura misurataPer i parametri morfologici polmonari ( es . MIL e ILV). I protocolli standardizzati possono essere utilizzati per ovviare a questa polarizzazione per determinare sia ILV che MLI, i due parametri utilizzati per il calcolo dell&#39;ISA. Qui forniamo protocolli altamente dettagliati e standardizzati per misurare questi parametri polmonari. Importante, la capacità di quantificare con precisione le promesse ISA migliora l&#39;affidabilità e la riproducibilità degli studi sulla funzionalità polmonare nei modelli di rigenerazione alveolare indotta da lesioni e dovrebbe facilitare le scoperte meccaniche in molteplici patologie polmonari.

<table><tbody><tr><td>Low cost cautery kit</td><td>Fine Science Tools</td><td>18010-00</td><td></td></tr><tr><td>Noyes scissors</td><td>Fine Science Tools</td><td>15012-12</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td></td></tr><tr><td>Castroviejo Micro Needle Holders</td><td>Fine Science Tools</td><td>12060-01</td><td></td></tr><tr><td>Vessel clips</td><td>Fine Science Tools</td><td>18374-44</td><td></td></tr><tr><td>I. V. Cannula-20 gauge</td><td>Jinhuan Medical Product Co., LTD.</td><td>29P0601</td><td></td></tr><tr><td>Surgical suture</td><td>Jinhuan Medical Product Co., LTD.</td><td>F602</td><td></td></tr><tr><td>Mouse intubation platform</td><td>Penn-Century, Inc</td><td>Model MIP</td><td></td></tr><tr><td>Small Animal Laryngoscope</td><td>Penn-Century, Inc</td><td>Model LS-2-M</td><td></td></tr><tr><td>TOPO Small Animal Ventilator</td><td>Kent Scientific</td><td>RSP1006-05L</td><td></td></tr><tr><td>Thermal pad</td><td>Stuart equipment</td><td>SBH130D</td><td></td></tr><tr><td>Pentobarbital sodium salt</td><td>Sigma</td><td>P3761</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Sigma</td><td>H3393</td><td></td></tr><tr><td>Hematoxylin Solution</td><td>Sigma</td><td>GHS132</td><td></td></tr><tr><td>Eosin Y solution, alcoholic</td><td>Sigma</td><td>HT110116</td><td></td></tr><tr><td>10 mL Pipette</td><td>Thermo Scientific</td><td>170356</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>O.C.T Compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>cryosection machine</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>Disposable Base Molds</td><td>Fisher HealthCare</td><td>22-363-553</td><td></td></tr><tr><td>18 gauge needle</td><td>Becton Dickinson</td><td>305199</td><td></td></tr><tr><td>Povidone iodine</td><td>Fisher Scientific</td><td>19-027132</td><td></td></tr><tr><td>70% ethanol</td><td>Fisher Scientific</td><td>BP82011</td><td></td></tr><tr><td>Infusion sets for single use</td><td>Weigao</td><td>SFDA 2012 3661704</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Tapes</td><td>3M Scotch</td><td>8915</td><td></td></tr><tr><td>Cotton pad</td><td>Vinda</td><td>Dr.P</td><td></td></tr><tr><td>Silicone prosthesis</td><td>Custom made</td><td></td><td></td></tr><tr><td>Brightfield microscope</td><td>Olympus</td><td>VS120</td><td></td></tr><tr><td>Ruler tool</td><td>Adobe Photoshop</td><td></td><td></td></tr></tbody></table>

a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

<html> Abbiamo eseguito qui un esperimento con un gruppo trattato con PNX e un gruppo di protesi (impianto protesico). Questi raggruppamenti sono gli stessi dei raggruppamenti utilizzati in uno studio precedentemente pubblicato dal nostro gruppo di ricerca 14 . Le mostre PNX e le protesi di protesi sono mostrate in Figura 2 . 8 settimane vecchi topi maschi CD-1 vengono utili...

Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

La superficie interna polmonare (ISA) è un criterio critico per valutare la morfologia polmonare e la fisiologia delle malattie polmonari e della rigenerazione alveolare indotta da lesioni. Qui descriviamo un metodo standardizzato che può ridurre al minimo la polarizzazione di misura per ISA sia nei modelli di polmonite polmonare sia nei modelli di protesi.

Un metodo standardizzato per la misurazione della superficie interna polmonare tramite la polmonite e l&#39;impianto di protesi

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

a-standardized-method-for-measuring-internal-lung-surface-area-via

Research

JoVE Journal

Developmental Biology

13.1K Views.  Peking University.  La superficie interna polmonare (ISA) è un criterio critico per valutare la morfologia polmonare e la fisiologia delle malattie polmonari e della rigenerazione alveolare indotta da lesioni. Qui descriviamo un metodo standardizzato che può ridurre al minimo la polarizzazione di misura per ISA sia nei modelli di polmonite polmonare sia nei modelli di protesi. 

Video: A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

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  ...

Ricerca

Medicina

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Nella misura in vivo della superficie polmonare strato endoteliale mouse

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Una finestra permanente per indagare sulle metastasi del cancro al polmone

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

Ricerca sul cancro

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Misurazione automatica di enfisema polmonare e piccole vie aeree ritocca in sigaretta Mice-fumo esposti

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

Topo Pneumonectomia Modello di crescita compensatoria Lung

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

Misura della curva di pressione-volume in mouse polmoni

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer research. Although there are a number of methods that have been published showing how to intubate mice for pulmonary function measurements, none are without potential problems for rapid tracheal instillation in large cohorts of mice. In the present paper, a simple and quick method is described that enables an investigator to carry out such instillations in an efficient manner. The method does not require any special tools or lighting and can be learned with very little practice. It involves anesthetizing a mouse, making a small incision in the neck to visualize the trachea, and then inserting an intravenous catheter directly. The small incision is quickly closed with tissue adhesive, and the mice are allowed to recover. A skilled student or technician can do instillations at an average rate of 2 min/mouse. Once the cancer is established, there is frequently a need for quantitative histologic analysis of the lungs. Traditionally pathologists usually do not bother to standardize lung inflation during fixation, and analyses are often based on a scoring system that can be quite subjective. While this may sometime be sufficiently adequate for gross estimates of the size of a lung tumor, any proper stereological quantification of lung structure or cells requires a reproducible fixation procedure and subsequent lung volume measurement. Here we describe simple reliable procedures for both fixing the lungs under pressure and then accurately measuring the fixed lung volume. The only requirement is a laboratory balance that is accurate over a range of 1 mg&#8211;300 g. The procedures presented here thus could greatly improve the ability to create, treat, and analyze lung cancers in mice.

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

Instillazione e fissaggio Metodi utili in mouse Lung Cancer Research

The ability to instill live agents, cells, or chemicals directly into the lung without injuring or killing the mice is an important tool in lung cancer ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Prova di tolleranza di glucosio intraperitoneale, misura della funzione polmonare e la fissazione del polmone per studiare l'impatto dell'obesità e metabolismo alterato sugli esiti polmonare

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Immunologia e infezioni

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Pre-condizionamento delle vie aeree dei topi con bleomicina aumenta l'efficienza di Orthotopic del polmone cancro Engraftment delle cellule

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

<meta charset="utf8"></head><body>Monitoraggio in tempo reale delle dinamiche cellulari nel trapianto di polmone di topo

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>Autore in primo piano: Avanzamento dell'immunologia dei trapianti di polmone attraverso l'imaging intravitale

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Microscopia intravitale a due fotoni del polmone di topo trapiantato

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

Un metodo standardizzato per la misurazione della superficie interna polmonare tramite la polmonite e l'impianto di protesi

Riepilogo

Esplora Altri Video

Capitoli in questo video