Wir möchten Francesca Polverino MD, wissenschaftlicher Mitarbeiter am Brigham and Women s Hospital für ihren Beitrag zu diesem Artikel danken und auch Monica Yao, BS, und Kate Rydell, BS für die Unterstützung bei murinen Haltung und Aussetzen der Mäuse gegenüber Zigarettenrauch. Diese Arbeit wurde vom Public Health Service, National Heart, Lung, and Blood Institute Grants HL111835, HL105339, HL114501, Flugbegleiter Medical Research Institute Grant # CIA123046, der Brigham und Krankenhaus-Lovelace Respiratory Research Institute Consortium Frauen und der Cambridge NIHR Biomedical unterstützt Forschungszentrum.

<ol>
	<li>Murray, C. J., Lopez, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+global+burden+of+disease.">Measuring the global burden of disease.</a> N. Engl. J Med. 369, 448-457 (2013).</li><li>Wright, J. L., Cosio, M., Churg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+chronic+obstructive+pulmonary+disease.">Animal models of chronic obstructive pulmonary disease.</a> Am. J Physiol Lung Cell Mol. Physiol. 295, 1-15 (2008).</li><li>Hautamaki, R. D., Kobayashi, D. K., Senior, R. M., Shapiro, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+macrophage+elastase+for+cigarette+smoke-induced+emphysema+in+mice.">Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice.</a> Science. 277, 2002-2004 (1997).</li><li>Churg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+intervention+with+a+myeloperoxidase+inhibitor+stops+progression+of+experimental+chronic+obstructive+pulmonary+disease.">Late intervention with a myeloperoxidase inhibitor stops progression of experimental chronic obstructive pulmonary disease.</a> Am. J. Respir. Crit Care Med. 185, 34-43 (2012).</li><li>Churg, A., Zhou, S., Wang, X., Wang, R., Wright, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+interleukin-1beta+in+murine+cigarette+smoke-induced+emphysema+and+small+airway+remodeling.">The role of interleukin-1beta in murine cigarette smoke-induced emphysema and small airway remodeling.</a> Am J Respir. Cell Mol. Biol. 40, 482-490 (2009).</li><li>Hogg, J. 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=The+nature+of+small-airway+obstruction+in+chronic+obstructive+pulmonary+disease.">The nature of small-airway obstruction in chronic obstructive pulmonary disease.</a> N. Engl. J. Med. 350, 2645-2653 (2004).</li><li>Paigen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miracle+enough:+the+power+of+mice.">A miracle enough: the power of mice.</a> Nat. Med. 1, 215-220 (1995).</li><li>Vlahos, R., Bozinovski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+pre-clinical+mouse+models+of+COPD.">Recent advances in pre-clinical mouse models of COPD.</a> Clin. Sci. (Lond). 126, 253-265 (2014).</li><li>Churg, A., Tai, H., Coulthard, T., Wang, R., Wright, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+drives+small+airway+remodeling+by+induction+of+growth+factors+in+the+airway+wall).">Cigarette smoke drives small airway remodeling by induction of growth factors in the airway wall).</a> Am. J. Respir. Crit Care Med. 174, 1327-1334 (2006).</li><li>Guerassimov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+emphysema+in+cigarette+smoke-exposed+mice+is+strain+dependent.">The development of emphysema in cigarette smoke-exposed mice is strain dependent.</a> Am. J. Respir. Crit Care Med. 170, 974-980 (2004).</li><li>van Eijl, S., van Oorschot, R., Olivier, B., Nijkamp , F. P., Bloksma, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+hypothermia+in+mice+in+a+nose-only+cigarette+smoke+exposure+system.">Stress and hypothermia in mice in a nose-only cigarette smoke exposure system.</a> Inhal. Toxicol. 18, 911-918 (2006).</li><li>Mauderly, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+3+methods+of+exposing+rats+to+cigarette+smoke.">Comparison of 3 methods of exposing rats to cigarette smoke.</a> Exp. Pathol. 37, 194-197 (1989).</li><li>Yao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+superoxide+dismutase+protects+against+pulmonary+emphysema+by+attenuating+oxidative+fragmentation+of+ECM.">Extracellular superoxide dismutase protects against pulmonary emphysema by attenuating oxidative fragmentation of ECM.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 15571-15576 (2010).</li><li>Crane-Godreau, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+influence+of+vitamin+D+deficiency+on+cigarette+smoke-induced+emphysema.">Modeling the influence of vitamin D deficiency on cigarette smoke-induced emphysema.</a> Front Physiol. 4, 132 (2013).</li><li>McGovern, T. K., Robichaud, A., Fereydoonzad, L., Schuessler, T. F., Martin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+respiratory+system+mechanics+in+mice+using+the+forced+oscillation+technique.">Evaluation of respiratory system mechanics in mice using the forced oscillation technique.</a> J Vis. Exp. , e50172 (2013).</li><li>De Vleeschauwer, S. 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=Repeated+invasive+lung+function+measurements+in+intubated+mice:+an+approach+for+longitudinal+lung+research.">Repeated invasive lung function measurements in intubated mice: an approach for longitudinal lung research.</a> Lab Anim. 45, 81-89 (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, 320-328 (1962).</li><li>Saetta, M., 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=Destructive+index:+a+measurement+of+lung+parenchymal+destruction+in+smokers.">Destructive index: a measurement of lung parenchymal destruction in smokers.</a> Am Rev. Respir. Dis. 131, 764-769 (1985).</li><li>Saito, K., Cagle, P., Berend, N., 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+'destructive+index'+in+nonemphysematous+and+emphysematous+lungs.+Morphologic+observations+and+correlation+with+function.">The 'destructive index' in nonemphysematous and emphysematous lungs. Morphologic observations and correlation with function.</a> Am Rev. Respir. Dis. 139, 308-312 (1989).</li><li>Robbesom, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+quantification+of+emphysema+in+small+human+lung+specimens:+comparison+of+methods+and+relation+with+clinical+data.">Morphological quantification of emphysema in small human lung specimens: comparison of methods and relation with clinical data.</a> Mod. Pathol. 16, 1-7 (2003).</li><li>Moghadaszadeh, B., 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=Selenoprotein+N+deficiency+in+mice+is+associated+with+abnormal+lung+development.">Selenoprotein N deficiency in mice is associated with abnormal lung development.</a> FASEB J. 4, 1585-1599 (2013).</li><li>Churg, A., Sin, D. D., Wright, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+prevents+emphysema:+are+animal+models+of+cigarette+smoke-induced+chronic+obstructive+pulmonary+disease+any+use.">Everything prevents emphysema: are animal models of cigarette smoke-induced chronic obstructive pulmonary disease any use.</a> Am J Respir. Cell Mol. Biol. 45, 1111-1115 (2011).</li><li>McComb, J. G., 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=CX3CL1+up-regulation+is+associated+with+recruitment+of+CX3CR1++mononuclear+phagocytes+and+T+lymphocytes+in+the+lungs+during+cigarette+smoke-induced+emphysema.">CX3CL1 up-regulation is associated with recruitment of CX3CR1+ mononuclear phagocytes and T lymphocytes in the lungs during cigarette smoke-induced emphysema.</a> Am. J. Pathol. 173, 949-961 (2008).</li><li>Mizumura, 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=Mitophagy-dependent+necroptosis+contributes+to+the+pathogenesis+of+COPD.">Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD.</a> J. Clin. Invest. 124, 3987-4003 (2014).</li></ol>

We have no conflicts of interest to disclose.

Most researcher use mice to model the main chronic lung pathologies and abnormal lung physiology in COPD (airspace enlargement, SAR, and increases in lung compliance) present in the human disease. A comprehensive approach to assess the effect of molecules of interest on both emphysema development and SAR is needed in mice in order to comprehensively assess the activities of molecules of interest in these chronic disease processes. 
There are several critical steps in this protocol. First, dur...

Die Verwendung von Tiermodellen zu untersuchen COPD ist schwierig, weil kein Modell perfekt zu allen Funktionen des menschlichen Krankheit (2) zu replizieren. Die meisten Forscher verwenden Mäuse COPD aufgrund der Ähnlichkeiten zwischen Mäusen und Menschen in ihrem Lungen Physiologie, Pathologie, Genetik und Metaboliten zu modellieren. Auch sind Mäuse relativ kostengünstig zu studieren, und beide Emphysem und kleinen Umbau der Atemwege entwickeln sich innerhalb von 6 Monaten nach CS Exposition (5,7-9). Zigarettenrauch induzierten COPD: Verschiedene Methoden können COPD in Mäusen zu induzieren. Die meisten Forscher entlarven Mäuse CS, die die Haupt ätiologischer Faktor für die menschliche COPD ist. CS Exposition für 6 Monate bewirkt, dass die Entwicklung von Emphysem und der kleinen Atem Remodellierung (SAR) in Mäusen, aber die Schwere der Krankheit, die induziert wird, variiert in Abhängigkeit von dem murinen Stamm untersucht. Zum Beispiel sind NZWLacZ Mäuse resistent gegen die Entwicklung von CS-induzierte Emphysem wohin AKR / J-Mäuse sind extremely empfindlich (10). Die meisten Forscher studieren C57BL / 6-Mäuse-Stamm in der CS-Expositionsmodell so viele Gen-Targeting-Mäuse sind in dieser Sorte zur Verfügung. Nach 6 Monaten der CS Exposition, Emphysem und der kleinen Atemwege Fibrose in Wildtyp zu entwickeln (WT) C57BL / 6 Mäusen, und beide Läsionen sind in der Schwere (5,10) relativ mild. Die Forscher nutzen zwei Arten von CS Exposition: Nase nur und Ganzkörperaufnahmen. Die Hauptnachteile der Nase nur Aufnahmetechnik sind, dass: 1) es ist ein arbeitsintensiver Verfahren ist; und 2) Mäuse müssen in kleine Kammern, die eine Stress-Reaktion und Hyperthermie in der Tiere (11) veranlassen können zurückgehalten werden. Der größte Nachteil der Ganzkörperexposition (hier beschrieben) ist, dass die Tiere sich nehmen (wie auch inhalieren) Nikotin und Teer-Produkte, wenn sie ihr Fell zu reinigen. Mäuse von Ganzkörper-CS ausgesetzt haben auch eine geringere Carboxyhämoglobin und ein geringeres Körpergewichtsverlust, wenn sie mit Tier Nasen nur CS (12) ausgesetzt ist, verglichen. Lungenfunktionsprüfung (PFT): Maßnahmen der Lungencompliance und Elastance sind in der Regel ähnlich in C57BL / 6-Wildtyp (WT) Mäusen aufgrund der relativ milden Emphysem, dass, wenn dieses entwickelt, um Luft oder CS für 6 Monate ausgesetzt Stamm auf CS (10) ausgesetzt ist. Wenn jedoch emphysematous Zerstörung schwerere, Erhöhung der Lungencompliance und Linksverschiebungen in der Druck-Volumen (PV) zu fließen, Schleifen detektiert werden kann. Letzteres kann beobachtet werden, beispielsweise in Mausstämme, die anfällig für die Auswirkungen von CS (10) sind, in CS-belichteten C57BL / 6-Stamm Gen-Targeting-Mäusen, die eine schwerere Emphysem Typ als C57BL / 6-Mäuse haben WT (13) oder in CS-exponierten Mäusen gegenüber Umweltveränderungen, die sie anfällig für die Auswirkungen von CS (14) zu machen unterzogen. Dieses Protokoll verwendet einen kleinen Tierbeatmungsgerät Rabatte der elastischen Rückstoß der Lunge (Anstieg der quasistatischen Lungencompliance [Cst] und Ermäßigungen in Gewebe zu messenElastanz [H]), PV Strömungsschleifen, und Veränderungen in den Atemwegen und Gewebefestigkeit bei anästhesierten Mäusen (15,16). Maßnahmen von Lungenemphysem: Analyse von Emphysem Entwicklung CS-exponierten C56BL / 6-Mäuse-Stamm ist eine Herausforderung, da ihre Verteilung ist räumlich heterogen. Mehrere verschiedene Methoden zu quantifizieren Luftraumvergrößerung bei Mäusen. Das erste Verfahren verwendet wurde, war die mittlere Linearabschnitt (L m) (17). Ist die L m Verfahren jedoch eine langsame, manuelle Prozess, der die Heterogenität der Erkrankung (es sei denn, alle Abschnitte der Lunge zufällig abgetastet) und seine Verwendung kann daher Beobachter Bias einführen in die Analyse nicht erfassen können. Die Zerstörungsindex [DI, (18)] quantifiziert auch Luftraum Erweiterung mit einem transparenten Blatt mit 50 gleichmäßig verteilten Punkten über einer gedruckten digitalisierten Bild einer Hämatoxylin und Eosin-gefärbte Lungenabschnitt platziert. Die PI-Verfahren erzielt das Gebiet um jeden Punkt acCording in dem Maße, in dem die Alveolargänge und Alveolarwände in diesem Bereich werden zerstört. Der Hauptnachteil des DI-Methode ist, dass es zeitaufwendig und genauer als andere Verfahren (19,20). Dieses Protokoll Maßnahmen bedeuten alveolären Sehnenlänge und Alveolarbereich auf Paraffin eingebettete Lungenschnitten mit Gills Fleck befleckt. Morphometrie Software wandelt Bilder von Lungenschnitten auf binäre Bilder (in dem Gewebe ist weiß und Luftraum ist schwarz), und dann überlagert ein einheitliches Raster aus horizontalen und vertikalen Linien (Akkorde) und die Software quantifiziert dann die Länge der einzelnen Saite in Gebieten identifiziert Software als Luftraum. Mit dieser Methode ist es möglich, die Größe der Alveolen in allen Teilen der Lunge in einer standardisierten und relativ automatisierte Weise (21) zu messen. Kleine Umbau der Atemwege (SAR): Die erhöhte Ablagerung von ECM-Proteinen (insbesondere interstitial Kollagene) um kleinen Atemwege erfolgt im CS-exponierten Tieren und trägt zu einer Behinderung des Luftstroms. Forscher nicht SAR im Tiermodell der COPD so oft wie Emphysem Entwicklung (22) zu studieren. SAR in CS-exponierten Mäusen quantifizieren, verwendet dieses Protokoll, Bildanalyse-Software, um die Dicke der Schicht aus ECM-Proteinen, die in der Umgebung der kleinen Atemwege (Atemwege mit einem mittleren Durchmesser zwischen 300 und 899 m) in Paraffin eingebetteten Lungenschnitten aufgebracht wird gemessen mit Masson-Trichrom-Färbung gefärbt.

<table border="0" cellpadding="0" cellspacing="0" width="579">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">Whole-body smoke exposure device</td>
			<td style="width:103px;">Teague Enterprise</td>
			<td style="width:101px;">TE-10z</td>
			<td rowspan="2" style="width:153px;">Chronic Smoke exposures to induce chronic lung disease in mice</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">Research Cigarette</td>
			<td style="width:103px;">University of Kentucky</td>
			<td style="width:101px;">3R4F reference cigarettes</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">Pallflex&#174; Air Monitoring Filters, Emfab Filters TX40HI20WW, 25 mm</td>
			<td style="width:103px;">Pall Corporation</td>
			<td style="width:101px;">7219</td>
			<td rowspan="4" style="width:153px;">For measurement of TPMs</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:223px;">25 mm filter holder</td>
			<td style="width:103px;">Pall Corporation</td>
			<td style="width:101px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:223px;">Filter sampler</td>
			<td style="width:103px;">Intermatic</td>
			<td style="width:101px;">Metal T100</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">Gas meter</td>
			<td style="width:103px;">AEM</td>
			<td style="width:101px;">Gas meters G1.6; G2.5; G4</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">Tracheal Cannula for mouse 18 gauge</td>
			<td style="width:103px;">Labinvention</td>
			<td style="width:101px;"></td>
			<td rowspan="2" style="width:153px;">Analysis of pulmonary function</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:223px;">Mechanical ventilator</td>
			<td style="width:103px;">Scireq</td>
			<td style="width:101px;">FlexiVent</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:223px;">Gill&#39;s hematoxylin solution&#160;</td>
			<td style="width:103px;">Sigma-Aldrich</td>
			<td style="width:101px;">GSH316</td>
			<td rowspan="3" style="width:153px;">For Gill staining, work under a fume hood</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">Hematoxylin solution, Harris modified</td>
			<td style="width:103px;">Sigma-Aldrich</td>
			<td style="width:101px;">HHS16</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">Cytoseal-60</td>
			<td style="width:103px;">Thermo Scientific</td>
			<td>8310-16</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">Micro-Slide-Field-Finder</td>
			<td style="width:103px;">Andwin Scientific INC</td>
			<td>50-949-582</td>
			<td rowspan="2" style="width:153px;">For analysis of emphysema</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">Scion Image Program</td>
			<td style="width:103px;">Scion Corporation</td>
			<td style="width:101px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:223px;">Mason&#39;s trichrome stain</td>
			<td style="width:103px;">Sigma-Aldrich</td>
			<td style="width:101px;">HT15</td>
			<td rowspan="2" style="width:153px;">For analysis of small airway fibrosis</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:223px;">MetaMorp Offline version 7.0</td>
			<td style="width:103px;">Molecullar Devices LLC</td>
			<td style="width:101px;">31032</td>
		</tr>
	</tbody>
</table>

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.

Dieses Protokoll beginnt mit Ganzkörper-Exposition von Mäusen für CS. Angemessene Aufsicht und Wartung des Gerätes und die Überwachung der TPM zählt eine konsistente Rauch Exposition (Abbildung 1). Es ist wichtig, dass der Forscher übt die Lunge Inflation Technik unter Verwendung der Aufblasvorrichtung Dieses Protokoll beginnt mit Ganzkörper-Exposition von Mäusen für CS. Angemessene Aufsicht und Wartung des Gerätes und die Überwachung der TPM zählt eine konsiste...

Watch this Scientific Journal Video about Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice at JoVE.com

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

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 ...

automated-measurement-pulmonary-emphysema-small-airway-remodeling

Research

JoVE Journal

Medicine

13.1K Aufrufe.  Brigham and Women's Hospital - Harvard Medical School. 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. 

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

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Isolierung von Maus respiratorischen Epithelzellen und der Exposition gegenüber Experimental Zigarettenrauch bei Air Liquid-Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Forschung

Medizin

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Protokoll für Long Duration Ganzkörper-Hyperthermie in Mäuse

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

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.

In vivo Messung der Maus 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 ...

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Echokardiographische Beurteilung des rechten Herzens bei Mäusen

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Automatisierte Messung der Mikrozirkulation Blutflußgeschwindigkeit in Lungenmetastasen von Ratten

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

An Improved Method for Rapid Intubation of the Trachea in Mice

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

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Ein verbessertes Verfahren zur schnellen Intubation der Luftröhre in Mice

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

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Ein Modell der kardialen Remodeling Durch Zusammenziehen der Bauchaorta bei Ratten

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

Vergleich der Effekte von elektronischen Zigarettendampf und Zigarettenrauch in einem Roman<em&gt; In vivo</em&gt; Belichtungssystem

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung fixation to produce reliable histological specimens of the lung. Due to the nature of the lung&rsquo;s structural composition, which consists largely of air and tissue, there is a risk that it collapses or deflates during the fixation process. Various lung fixation methods exist, each of which has its own advantages and disadvantages. The lung fixation method presented here utilizes constant pressure to enable optimal tissue evaluation for studies using an emphysematous mouse lung model. The main advantage is that it can fix many lungs with the same condition at one time. Lung specimens are obtained from chronic cigarette smoke-exposed mice. Lung fixation is performed using specialized equipment that enables the production of constant pressure. This constant pressure maintains the lung in a reasonably inflated state. Thus, this method generates a histological specimen of the lung that is suitable to evaluate cigarette smoke-induced mild emphysema.

Presented here is a useful protocol for lung fixation that creates a stable condition for histological evaluate of lung specimens from a mouse model of emphysema. The main advantage of this model is that it can fix many lungs with the same constant pressure without lung collapse or deflation.

Lungenfixierung unter konstantem Druck zur Beurteilung von Emphysem bei Mäusen

Emphysema is a significant feature of chronic obstructive pulmonary disease (COPD). Studies involving an emphysematous mouse model require optimal lung ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

Transdermale Messung der glomerulären Filtrationsrate bei Mäusen

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...