Name: phenotyping mouse pulmonary function in vivo with the lung diffusing capacity
Uploaded: 2015-01-06T10:00:00.000+00:00
Duration: 7 min 13 s
Description: Watch this Scientific Journal Video about Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity at JoVE.com

This work was supported by NIH HL-10342

HINWEIS: Alle Tier Protokolle wurden von der Johns Hopkins University Animal Care und Verwenden Committee genehmigt. 1. Vorbereitung der Tiere <ol><li> Bereiten Sie 6 C57BL / 6-Kontrollmäuse für die DFCO Messung von betäuben sie mit Ketamin und Xylazin wie in Schritt 2.3 unten beschrieben. </li><li> Bereiten alle anderen Mäuse mit den in Tabelle 1 gezeigten verschiedenen Lungenerkrankungen durch Verwendung des gleichen Verfahrens wie für die Kontrollen. Spezifische Details benötigt, um jedes dieser Modelle zu etablieren sind in den entsprechenden Referenzen gefunden. Die Kontrollmäuse und die in den anderen pathologischen Kohorten alle 6-12 Wochen alt. </li></ol> 2. Messung der Diffusion Faktor für Kohlenmonoxid (DFCO) <ol><li> Einrichten des Gaschromatographen Modul mit der Maschine zugeführt, um Peaks für Stickstoff, Sauerstoff, Neon und Kohlenmonoxid zu messen. Für diese Anwendung verwenden nur die Neon und CO-Daten. HINWEIS: Dieses Gerät verwendet eine molekulare sIEVE Säule mit Helium als Trägergas mit einem 12,00 um Film, 320,00 &amp; mgr; m-ID und 10 m Länge. Der Chromatograph Säule mit einem Volumen von 0,8 ml, aber wir 2 ml ausreichende Lichtung der Verbindungsschlauch mit der Probe zu gewährleisten. </li><li> Zu Beginn eines jeden Versuches Tag, vor der Durchführung von Messungen von Proben, die von Mäusen, einen 2 ml Probe direkt aus einem Gasgemisch enthaltenden Beutel ungefähr 0,5% Ne, 0,5% CO, Rest Luft, und diese Probe zu kalibrieren Gaschromatograph. </li><li> Anesthetize Mäuse mit Ketamin (90 mg / kg) und Xylazin (15 mg / kg), und bestätigen Narkose durch das Fehlen von Rückbewegung. Bewerben Tier Salbe auf die Augen bis zur Trockenheit zu verhindern. Tracheostomize die Mäuse mit einer Stichnadelkanüle (18 G bei Erwachsenen bzw. 20 G bei sehr jungen Mäuse). HINWEIS: Die DFCO wird in weniger als 10 min nach der Narkose beendet und vor jeder mechanischen Ventilation oder andere Verfahren. </li><li> Bei Mäusen mehr als 6 Wochen alt, mit einem 3-ml-Spritze to zurückzuziehen 0,8 ml Gas aus dem Gasgemisch Beutel. Verbinden Sie die Spritze mit der Trachealkanüle und schnell aufblasen die Lunge. Mit Hilfe eines Metronoms, zählen 9 sec, und dann schnell ziehen Sie die 0,8 ml (ausgeatmete Luft). </li><li> Verdünnen Sie diese zurückgezogen 0,8 ml ausgeatmete Luft auf 2 ml mit Raumluft, lassen Sie es für mindestens 15 Sekunden Ruhe. Dann injizieren die gesamte Probe in den Gaschromatographen für die Analyse. </li><li> Bei der Analyse dieses ersten DFCO Probe, pumpen Sie die Mauslunge mit einem zweiten 0,8 ml aus dem Gasgemisch Tasche, und dann verarbeiten dieses Beispiel identisch mit der ersten Probe. Der Mittelwert der beiden Messungen DFCO. HINWEIS: Für die Messung in Mäusen im Alter von 2 Wochen alt, mit einem Volumen von 0,4 ml, 0,8 ml, da ist das umfangreiche Messungen in Lungen von sehr jungen Mäusen zu machen. Es ist besser, das 0,8 ml Volumen für Mäuse älter als 6 Wochen verwendet, und dass, wenn der 0,4-ml-Volumen bei einigen Mäusen benötigt wird, es wird durchgehend für alle Mäuse der Kohorten untersucht werden. </li><li> Berechnen DFCOB. 1 - (CO 9 / CO c) / (Ne 9 / Ne c), wobei c und 9 Indices beziehen sich auf Konzentrationen der Kalibrierungsgase eingespritzt und die Gase nach einer 9 sec Atemanhaltezeit entfernt sind. </li><li> Analyse und den Vergleich Unterschiede mit einem one-way ANOVA und die Bedeutung Ebene mit Tukey-Korrektur für Mehrfachvergleiche in allen Kohorten-Mäusen. Betrachten p &lt;0,05 als signifikant Wert. HINWEIS: Alle hier verwendeten Mäuse waren Teil der experimentellen Studien mit mehreren aufeinander folgenden Messungen der Lungenventilation, Mechanik, Lungenspülung oder Histologie, der hier nicht ausgewiesen werden. Da zusätzlich das Verfahren ist das gleiche in allen experimentellen Modellen, wie oben bei den Kontrollmäusen durchgeführt wurde, werden nur die Ergebnisse von den verschiedenen pathologischen Modellen vorgestellt. Weitere Informationen zu diesen Modellen wird in der zusätzlichen Tabelle dargestellt. </li><li> Euthanize die Tiere durch tiefe anesthetic Überdosierung durch Genickbruch folgte oder Enthauptung. Wo nötig, entfernen Sie die Lungenzellen und / oder Geweben von den toten Mäusen für die weitere biologische oder histologische Verarbeitung und Analyse. </li></ol>

<ol>
	<li>Ogilvie, C. M., Forster, R. E., Blakemore, W. S., Morton, J. 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+standardized+breath+holding+technique+for+the+clinical+measurement+of+the+diffusing+capacity+of+the+lung+for+carbon+monoxide.">A standardized breath holding technique for the clinical measurement of the diffusing capacity of the lung for carbon monoxide.</a> J Clin Invest. 36 (1 Pt 1), 1-17 (1957).</li><li>Miller, A., Warshaw, R., Nezamis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusing+capacity+and+forced+vital+capacity+in+5,003+asbestos-exposed+workers:+Relationships+to+interstitial+fibrosis+(ILO+profusion+score)+and+pleural+thickening.">Diffusing capacity and forced vital capacity in 5,003 asbestos-exposed workers: Relationships to interstitial fibrosis (ILO profusion score) and pleural thickening.</a> Am J Ind Med. 56 (12), 1383-1393 (2013).</li><li>Enelow, R. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+consequences+of+alveolar+cell+recognition+by+CD8(+)+T+lymphocytes+in+experimental+lung+disease.">Structural and functional consequences of alveolar cell recognition by CD8(+) T lymphocytes in experimental lung disease.</a> J Clin Invest. 102 (9), 1653-1661 (1998).</li><li>Hartsfield, C. L., Lipke, D., Lai, Y. L., Cohen, D. A., Gillespie, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+mechanical+and+immunologic+dysfunction+in+a+murine+model+of+AIDS.">Pulmonary mechanical and immunologic dysfunction in a murine model of AIDS.</a> Am J Physiol. 272 (4 Pt 1), 699-706 (1997).</li><li>Wegner, C. D., 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=Intercellular+adhesion+molecule-1+contributes+to+pulmonary+oxygen+toxicity+in+mice:+role+of+leukocytes+revised.">Intercellular adhesion molecule-1 contributes to pulmonary oxygen toxicity in mice: role of leukocytes revised.</a> Lung. 170 (5), 267-279 (1992).</li><li>Reinhard, 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=Inbred+strain+variation+in+lung+function.">Inbred strain variation in lung function.</a> Mamm Genome. 13 (8), 429-437 (2002).</li><li>Sabo, J. P., Kimmel, E. C., Diamond, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+Clara+cell+toxin,+4-ipomeanol,+on+pulmonary+function+in+rats.">Effects of the Clara cell toxin, 4-ipomeanol, on pulmonary function in rats.</a> J Appl Physiol. 54 (2), 337-344 (1983).</li><li>Depledge, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiration+and+lung+function+in+the+mouse,+Mus+musculus+(with+a+note+on+mass+exponents+and+respiratory+variables).">Respiration and lung function in the mouse, Mus musculus (with a note on mass exponents and respiratory variables).</a> Respir Physiol. 60 (1), 83-94 (1985).</li><li>Depledge, M. H., Collis, C. H., Barrett, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+measuring+carbon+monoxide+uptake+in+mice.">A technique for measuring carbon monoxide uptake in mice.</a> Int J Radiat Oncol Biol Phys. 7 (4), 485-489 (1981).</li><li>Fallica, J., Das, S., Horton, M. R., 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=Application+of+Carbon+Monoxide+Diffusing+Capacity+in+the+Mouse+Lung.">Application of Carbon Monoxide Diffusing Capacity in the Mouse Lung.</a> J Appl Physiol. 110 (5), 1455-1459 (2011).</li><li>Chaudhary, N., Datta, K., Askin, F. B., Staab, J. F., Marr, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+transmembrane+conductance+regulator+regulates+epithelial+cell+response+to+Aspergillus+and+resultant+pulmonary+inflammation.">Cystic fibrosis transmembrane conductance regulator regulates epithelial cell response to Aspergillus and resultant pulmonary inflammation.</a> Am J Respir Crit Care Med. 185 (3), 301-310 (2012).</li><li>Foster, W. M., Walters, D. M., Longphre, M., Macri, K., Miller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+the+measurement+of+mucociliary+function+in+the+mouse+by+scintigraphy.">Methodology for the measurement of mucociliary function in the mouse by scintigraphy.</a> J Appl Physiol. 90 (3), 1111-1117 (2001).</li><li>Yildirim, A. O., 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=Palifermin+induces+alveolar+maintenance+programs+in+emphysematous+mice.">Palifermin induces alveolar maintenance programs in emphysematous mice.</a> Am J Respir Crit Care Med. 181 (7), 705-717 (2010).</li><li>Collins, S. L., Chan-Li, Y., Hallowell, R. W., Powell, J. D., Horton, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vaccination+as+a+novel+treatment+for+lung+fibrosis.">Pulmonary vaccination as a novel treatment for lung fibrosis.</a> PLoS One. 7 (2), e31299 (2012).</li><li>Alessio, F. 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=CD4+CD25+Foxp3++Tregs+resolve+experimental+lung+injury+in+mice+and+are+present+in+humans+with+acute+lung+injury.">CD4+CD25+Foxp3+ Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury.</a> J Clin Invest. 119 (10), 2898-2913 (2009).</li><li>Martinez, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+course+of+patients+with+idiopathic+pulmonary+fibrosis.">The clinical course of patients with idiopathic pulmonary fibrosis.</a> Ann Intern Med. 142 (12 Pt 1), 963-967 (2005).</li><li>Zhou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+lethal+intestinal+defect+in+a+mouse+model+of+cystic+fibrosis+by+human+CFTR.">Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR.</a> Science. 266 (5191), 1705-1708 (1994).</li></ol>

Interessenkonflikte, und nichts zu offenbaren.

In der vorliegenden Arbeit, definiert man eine neue Metrik, um die Gasaustauschfähigkeit der Mauslunge quantifizieren. Diese Metrik ist analog zu der Streukapazität eines gemeinsamen klinischen Messungen, die die primäre Funktion der Lunge misst, das heißt, seine Fähigkeit, den Gasaustausch. Die Diffusionskapazität ist die einzige Lungenfunktionsmessung, die leicht und schnell sowohl in Mäusen und Menschen durchgeführt werden können. Für den Nachweis von Lungenerkrankung bei Mäusen ist ein Hauptziel, um Lunge...

Die Maus ist nun der primäre Tier zur Behandlung einer Vielzahl von Lungenkrankheiten zu modellieren. Um die Mechanismen, die solche Krankheiten zugrunde liegen zu untersuchen, werden phänotypische Methoden benötigt, die es zu quantifizieren, können die pathologischen Veränderungen. Obwohl es viele Studien an Mäusen, wo Belüftungsmechanik gemessen werden, sind diese Messungen in der Regel nicht mit den Standarduntersuchungen zur Lungenfunktions normalerweise beim Menschen durchgeführt. Dies ist bedauerlich, da die Fähigkeit, gleichwertige Messungen bei Mäusen und menschlichen Probanden durchführen kann die Umsetzung der Ergebnisse in Mausmodellen für die menschliche Krankheit ermöglichen. Eine der häufigsten und leicht vorgenommen Messungen am Menschen ist die Diffusionskapazität für Kohlenmonoxid (DLCO) 1,2, aber diese Messung ist nur selten in Maus-Modellen geschehen. In diesen Studien, bei denen es wurde berichtet, 3-7, gab es keine Folgestudien, zum Teil, weil die Verfahren oft umständlich oder require komplexen Anlagen. Ein anderer Ansatz ist es, eine CO Rückatemmethode in einem stabilen Zustand System, das den Vorteil, dass sie in der Lage, CO-Diffusion in der bewussten Mäusen messen verfügt. Dieses Verfahren ist jedoch sehr aufwendig, und die Ergebnisse können mit dem Niveau der der Maus die Belüftung sowie O 2 und CO 2 -Konzentrationen 8,9 variieren. Diese Schwierigkeiten scheinen routinemäßige Anwendung von Diffusionskapazität der Lunge Pathologie bei Mäusen erkennen ausgeschlossen haben, trotz seiner mehrere Vorteile. Um die Probleme mit der Messung der Diffusionskapazität in Mäusen zu umgehen, Details der ein einfaches Mittel, um es in Mäusen gemessen wurden kürzlich berichtet 10. Das Verfahren schließt das schwierige Problem der Probenahme unberührten Alveolargas durch schnelles Abtasten eines Volumens gleich der gesamten Inspirationsgas. Dieses Verfahren führt zu einer sehr reproduzierbare Messung, der sogenannten Diffusionsfaktor für Kohlenmonoxid (DFCO), die empfindlich gegenüber einer Vielzahl von pathologic Veränderungen der Lunge Phänotyp. Die DFCO errechnet sich somit 1 - (CO 9 / CO c) / (Ne 9 / Ne c), wo die c und 9 Indices beziehen sich auf Konzentrationen der Kalibrierungsgase eingespritzt und die Gase nach einer 9 sec Atemanhaltezeit beseitigt, jeweils. DFCO ist eine dimensionslose Größe, die zwischen 0 und 1 variiert, wobei 1 reflektiert vollständige Aufnahme aller CO und 0 reflektierenden keine CO-Aufnahme. In diesem Vortrag zeigen wir, wie man diese Diffusionskapazität Messung durchzuführen, und wie sie verwendet werden, um Veränderungen in fast allen der vorhandenen Maus Lungenkrankheit Modelle, einschließlich Emphysem, Fibrose, akuter Lungenverletzung und Virus- und Pilzinfektionen zu dokumentieren.

<table><tbody><tr><td>Gas Chromatograph</td><td>Inficon</td><td>Micro GC Model 3000A</td><td>Agilent makes a comparable model</td></tr><tr><td>18 G Luer stub needle</td><td>Becton Dickenson</td><td></td><td>Several other possible vendors</td></tr><tr><td>3 ml plastic syringe</td><td>Becton Dickenson</td><td></td><td>Several other possible vendors</td></tr><tr><td>Polypropylene gas sample bags</td><td>SKC</td><td>1 or 2 L capacity works well</td><td>Other gas tight bags will work well</td></tr><tr><td>Gas tank, 0.3% Ne, 0.3% CO, balance air; (size ME)</td><td>Airgas, Inc</td><td>Z04 NI785ME3012</td><td>This is the standard mixture used for DLCO in humans</td></tr><tr><td>25 TCID50/mouse of influenza virus A/PR8 diluted in phosphate buffered saline.</td><td></td><td></td><td></td></tr><tr><td>Porcine pancreatic elastase</td><td>Elastin Products, Owensville, MO</td><td></td><td>5.4 U</td></tr><tr><td>Bleomycin</td><td>APP Pharmaceuticals, Schaumburg, IL</td><td></td><td>0.25 U</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; LPS</td><td>Sigma L2880</td><td></td><td>3 &amp;mu;g/g body weight; O55:B5</td></tr><tr><td>&lt;i&gt;Aspergillus fumigatus &lt;/i&gt; (isolate Af293) conidia were collected from mature colonies grown on potato dextrose agar.</td><td></td><td></td><td></td></tr></tbody></table>

phenotyping mouse pulmonary function in vivo with the lung diffusing capacity

Die Maus ist nun der primäre Tier zur Behandlung einer Vielzahl von Lungenkrankheiten zu modellieren. Um die Mechanismen, die solche Krankheiten zugrunde liegen, zu untersuchen, werden phänotypische Methoden benötigt, die pathologischen Veränderungen zu quantifizieren kann. Ferner, um eine Translations relevant Mausmodelle, sollten solche Messungen Tests, die leicht in Menschen und Mäusen durchgeführt werden kann. Leider ist in der vorliegenden Literatur einige phänotypische Messungen der Lungenfunktion haben die direkte Anwendung auf den Menschen. Eine Ausnahme ist die Diffusionskapazität für Kohlenmonoxid, das eine Messung, die routinemäßig in Menschen durchgeführt wird, ist. In diesem Bericht beschreiben wir ein Mittel, um schnell und einfach zu messen, diese Streukapazitäten bei Mäusen. Das Verfahren beinhaltet einen kurzen Lungeninflation mit Spürgase in anästhesierten Maus, gefolgt von einer 1-minütigen Gasanalysezeit. Wir haben die Fähigkeit dieses Verfahrens, mehrere Lungenerkrankungen, einschließlich Emphysem, Fibrose, akuter Lungenverletzung und Influenza und detektieren getestetPilz-Infektionen der Lunge, sowie die Überwachung Lungenreifung bei jungen Welpen. Die Ergebnisse zeigen eine signifikante Abnahme in allen Lungenerkrankungen, wie auch eine Erhöhung der Streukapazität bei Lungenreifung. Diese Messung der Lungendiffusionskapazität liefert somit eine Lungenfunktion, die eine breite Anwendung mit seiner Fähigkeit, phänotypische Strukturänderungen mit den meisten der vorhandenen pathologischen Lungen Modelle erfassen hat.

Abbildung 1 zeigt die DFCO Messungen von den erwachsenen Mäusen in den Gruppen A, B, C, D, E und F. Es wurden signifikante Abnahmen sowohl mit den Aspergillus und Influenza-Infektionen, sowie eine signifikante Abnahme in der fibrotischen, Emphysem, akute Lungenschädigung Modelle. Abbildung 2 zeigt die Gruppe G entwicklungsbedingten Veränderungen in DFCO im Laufe der Zeit als die Mäuse im Alter von 2-6 Wochen. Es gab eine kleine, aber signifikante Zunahme mit Lungenentwicklung in die...

Watch this Scientific Journal Video about Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity at JoVE.com

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

Phänotypisierung Maus Lungenfunktion<em&gt; In-vivo-</em&gt; Mit der Lungendiffusionskapazität

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

phenotyping-mouse-pulmonary-function-vivo-with-lung-diffusing

Research

JoVE Journal

Biology

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

10.5K Aufrufe.  Johns Hopkins University Bloomberg School of Public Health. We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

Serie: Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

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.

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

Eine standardisierte Methode zur Messung der inneren Lungenoberfläche über Maus Pneumonektomie und Prothesenimplantation

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

Forschung

Entwicklungsbiologie

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.

Automatisierte Messung von Lungenemphysem und Klein Airway Remodeling im Zigarettenrauch ausgesetzten Mäuse

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 ...

Medizin

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.

Die Messung der Druck-Volumen-Kurve in der Mäuselunge

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 ...

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.

Intraperitoneale Glucose-Toleranz-Test, Messung der Lungenfunktion und Fixierung der Lunge, um die Auswirkungen von Übergewicht und beeinträchtigter Stoffwechsel auf pulmonale Ergebnisse studieren

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

Immunologie und Infektion

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

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

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

Generation von eine chronisch obstruktive Lungenerkrankung-Modell bei Mäusen durch wiederholte Ozonbelastung

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

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

Auswertung der Atemwege Mechanik in Mäuse mit den Forced Oscillation Technique

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Biologie

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

Beurteilung der Atemfunktion in bewusste Mäuse durch Doppel-Kammer-Plethysmographie

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Hyperpolarized 129Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can be exploited to quantify pulmonary physiology, including ventilation, restricted diffusion (alveolar-airspace size), and gas exchange. In humans, it has been used to evaluate disease severity and progression in a variety of pulmonary disorders and is approved for clinical use in the United States and United Kingdom. Beyond its clinical applications, the ability of 129Xe MRI to noninvasively assess pulmonary pathophysiology and provide spatially resolved information is valuable for preclinical research. Among animal models, mice are the most widely used due to the accessibility of genetically modified disease models. Here, 129Xe MRI is promising as a minimally invasive, radiation-free, and sensitive technique to longitudinally monitor lung disease progression and therapy response (e.g., in drug discovery). This technique can extend to preclinical applications by incorporating an MRI-triggered, free-breathing apparatus or mechanical ventilator to deliver gas. Here, we describe the steps and provide checklists to ensure robust data collection and analysis, including creating a thermally polarized xenon gas phantom for quality control, optimizing polarization, animal handling (sedation, intubation, ventilation, and care for mice), and protocols for ventilation, restricted diffusion, and gas exchange data. While preclinical 129Xe MRI can be applied in various animal models (e.g., rats, pigs, sheep), this protocol focuses on mice due to the challenges posed by their small anatomy, which are balanced by their affordability and the availability of many disease models.

Hyperpolarized xenon MRI can quantify regional lung microstructure (air-space dimensions) and physiology (ventilation and gas exchange) in translational research and clinical care. Although challenging, it can provide comparable pulmonary insights in preclinical studies. This protocol describes the infrastructure and procedures needed to perform routine xenon lung MRI in mice.

Hyperpolarized (HP) xenon-129 (129Xe) is an inhaled magnetic resonance imaging (MRI) contrast agent with unique spectral and physical properties that can ...

Biotechnik

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Messatemfunktion im Maus Mit Hemmungslose Ganzkörper-Plethysmographie

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

In vivo Evaluation of Mucociliary Clearance in Mice

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a metachronal, synchronal fashion, these multiple, motile, actin-based organelles generate a cephalad fluid flow clearing the respiratory tract of inhaled pollutants and pathogens. With increasing environmental pollution, novel viral pathogens and emerging multi-drug resistant bacteria, cilia generated mucociliary clearance (MCC) is essential for maintaining lung health. MCC is also depressed in multiple congenital disorders like primary ciliary dyskinesia, cystic fibrosis as well as acquired disorders like chronic obstructive pulmonary disease. All these disorders have established, in some case multiple, mouse models. In this publication, we detail a method using a small amount of radioactivity and dual-modality SPECT/CT imaging to accurately and reproducibly measure MCC in mice in vivo. The method allows for recovery of mice after imaging, making serial measurements possible, and testing potential therapeutics longitudinally over time. The data in wild-type mice demonstrates the reproducibility of the MCC measurement as long as adequate attention to detail is paid, and the protocol strictly adhered to.

In this publication, we describe protocols for assessing airway mucociliary clearance (MCC) in mice in vivo utilizing dual-modality radionuclide imaging. This protocol is designed for a single photon emission computed tomography (SPECT) and computed tomography (CT) acquisition protocol using mouse whole body (MWB) collimators in a dual SPECT/CT system.

In vivo Bewertung der mukociliären Clearance bei Mäusen

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...

Phänotypisierung Maus Lungenfunktion

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video