Name: automated measurement of pulmonary emphysema and small airway remodeling in cigarette smoke-exposed mice
Uploaded: 2015-01-16T14:00:00
Description: Watch this Scientific Journal Video about Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice at JoVE.com

Wij willen Francesca Polverino MD, een Research Fellow aan de Brigham and Women&#39;s Hospital bedanken voor haar bijdrage aan dit artikel, en ook Monica Yao, BS, en Kate Rydell, BS voor hun hulp met muizen veeteelt en het blootstellen van de muizen aan sigarettenrook. Dit werk werd ondersteund door de GGD, National Heart, Lung, and Blood Institute Subsidies HL111835, HL105339, HL114501, Stewarden Medical Research Institute Grant # CIA123046, het Brigham and Women&#39;s Hospital-Lovelace Respiratory Research Institute Consortium, en de Cambridge NIHR Biomedische Centrum voor Onderzoek.

Het protocol neemt ~ 25 weken in beslag. Het protocol muizen blootgesteld aan lucht of rook voor 24 weken. Aan het einde van de rook posities, worden de protocol maatregelen longfunctie in de muizen, en de longen opgeblazen tot een vaste druk, gefixeerd en verwijderd op dezelfde dag. Meer tijd nodig is voor de onderzoeker te verankeren, te snijden, en vlekken op de longcoupes (2-3 dagen), en vast te leggen en het analyseren van beelden (2-4 dagen, afhankelijk van het aantal dieren bestudeerde). Dit protocol kan ook gebruikt worden om leefti...

<ol>
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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...

Het gebruik van diermodellen COPD studie is uitdaging omdat geen model perfect repliceren alle kenmerken van de menselijke ziekte (2). De meeste onderzoekers gebruiken muizen model COPD vanwege de overeenkomsten tussen muizen en mensen in de pulmonale fysiologie, pathologie, genetica en metabolieten. Ook muizen zijn relatief goedkoop om te studeren, en zowel longemfyseem en kleine luchtwegremodelling ontwikkelen binnen 6 maanden van CS blootstelling (5,7-9). Sigarettenrook geïnduceerde COPD: Verschillende methoden kunnen COPD induceren in muizen. De meeste onderzoekers blootstellen muizen CS, die de voornaamste etiologische factor voor menselijke COPD. CS blootstelling gedurende 6 maanden leidt de ontwikkeling van emfyseem en kleine luchtwegremodellering (SAR) bij muizen, maar de ernst van de ziekte die wordt geïnduceerd afhankelijk van het muizen stam onderzocht. Bijvoorbeeld, NZWLacZ muizen zijn bestand tegen de ontwikkeling van CS-geïnduceerd emfyseem dat AKR / J muizen extremely gevoelige (10). De meeste onderzoekers bestuderen C57BL / 6 muizen muizen in de CS blootstellingsmodel zoveel van transgene muizen in deze stam. Na 6 maanden CS blootstelling, emfyseem en kleine luchtwegen fibrose ontwikkelen wildtype (WT) C57BL / 6 muizen, en beide laesies zijn relatief mild van aard (5,10). De onderzoekers maken gebruik van twee soorten CS blootstelling: neus-only en het hele lichaam blootstellingen. De belangrijkste nadelen van de neus alleen belichtingstechniek zijn dat: 1) het is een arbeidsintensieve methode; en 2) muizen worden tegengehouden in kleine cellen die een stressrespons en hyperthermie in de dieren (11) kan induceren. Het grote nadeel van blootstelling gehele lichaam (hierin beschreven) dat de dieren kunnen innemen (evenals inademen) nicotine en teer producten wanneer ze schoon hun vacht. Muizen blootgesteld aan lichaamstrillingen CS hebben een lager carboxyhemoglobine niveaus en een vermindering van verlies van lichaamsgewicht in vergelijking met dieren blootgesteld aan neus slechts CS (12). longfunctietest (PFT): Handelingen longflexibiliteit en elasticiteit zijn gewoonlijk soortgelijk in C57BL / 6 wild type (WT) muizen blootgesteld aan lucht of CS gedurende 6 maanden als gevolg van de relatief milde emfyseem dat wanneer deze zich ontwikkelt stam wordt blootgesteld aan CS (10). Echter, wanneer emfysemateus vernietiging ernstiger toename longflexibiliteit en links verschuivingen in de druk-volume (PV) stroom lussen worden gedetecteerd. De laatste kan worden waargenomen, bijvoorbeeld in muizenstammen die meer vatbaar voor de effecten van CS (10), CS-blootgestelde C57BL / 6 stam van transgene muizen die een meer ernstige vorm emfyseem dan C57BL / 6 muizen WT (13), of CS-blootgestelde muizen blootgesteld aan omgevingsveranderingen dat ze meer vatbaar voor de effecten van CS (14) maken. Dit protocol maakt gebruik van een klein dier ventilator tot een vermindering van de elastische terugslag van de long (verhogingen van de quasi-statische longcompliantie [Constante] en verminderingen in het weefsel te metenelastantie [H]), PV stroom loops, en veranderingen in de luchtwegen en weefsel weerstand in verdoofde muizen (15,16). Maatregelen van longemfyseem: Analyse van emfyseem ontwikkeling in CS-blootgestelde C56BL / 6 stam muizen is uitdagend omdat de distributie is ruimtelijk heterogeen. Verschillende methoden kwantificeren luchtruim uitbreiding in muizen. De eerste methode was de gemiddelde lineaire intercept (L m) (17). De L m methode is een langzaam, handmatig proces die de heterogeniteit van de ziekte (tenzij alle delen van de long willekeurig bemonsterd) en het gebruik ervan kan derhalve voor de waarnemer voorspanning in de analyse kan vangen. De destructieve index [DI, (18)] kwantificeert ook het luchtruim uitbreiding met behulp van een transparant vel met 50 gelijk verdeeld punten geplaatst over een gedrukte gedigitaliseerde beeld van een hematoxyline en eosine gekleurd long sectie. De PI-methode scoort het gebied rondom elk punt acgens de mate waarin de alveolaire leidingen en alveolaire wanden binnen dit gebied worden vernietigd. Het belangrijkste nadeel van de DI methode is dat het tijdrovend en niet nauwkeuriger dan andere methoden (19,20). Dit protocol maatregelen betekenen alveolaire koordelengte en alveolaire gebied op paraffine ingebedde longcoupes gekleurd met Gill&#39;s vlek. Morfometrie software zet afbeeldingen longsecties voor binaire afbeeldingen (waarin weefsel wit en luchtruim zwart) en superponeert een uniform raster van horizontale en verticale lijnen (akkoorden) en de software kwantificeert dan de lengte van elk akkoord in gebieden die door software als het luchtruim. Met deze methode is het mogelijk om de grootte van de alveoli in alle delen van de long in een gestandaardiseerde en relatief geautomatiseerde wijze (21) te meten. Kleine luchtwegremodelling (SAR): De toegenomen afzetting van ECM eiwitten (vooral interstitial collagenen) rond kleine luchtwegen optreedt in CS-blootgestelde dieren en draagt ​​obstructie luchtstroom. Onderzoekers niet bestuderen SAR in diermodellen van COPD zo vaak als emfyseem ontwikkeling (22). Om SAR kwantificeren CS-blootgestelde muizen gebruikt dit protocol beeldanalyse software om de dikte van de laag van ECM eiwitten die wordt afgezet rond de kleine luchtwegen (luchtwegen met een gemiddelde diameter tussen 300 en 899 m) in paraffine ingebedde longsecties meten gekleurd met Masson trichroomkleuring vlek.

<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.

Dit protocol begint met hele lichaam blootstelling van muizen CS. Adequaat toezicht en het onderhoud van het apparaat en de monitoring van TBM telt zorgen voor een consistente rook blootstelling (figuur 1). Belangrijk is dat de onderzoeker oefent de longinflatie techniek met de opblaasinrichting Dit protocol begint met hele lichaam blootstelling van muizen CS. Adequaat toezicht en het onderhoud van het apparaat en de monitoring van TBM telt zorgen voor een consistente rook b...

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 (Video) | JoVE

Geautomatiseerde Meting van longemfyseem en Kleine luchtweg remodeling in sigarettenrook blootgesteld Muizen

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.

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 ...

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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  

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface (Video) | JoVE

Isolatie van Mouse Respiratory epitheelcellen en Blootstelling aan Experimentele Cigarette Smoke bij 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 ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Protocol for Long Duration Whole Body Hyperthermia in Mice (Video) | JoVE

Protocol voor Long Duration Whole Body Hyperthermie in Muizen

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

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer (Video) | JoVE

In vivo meting van de muis Pulmonary Endothelial oppervlaktelaag

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

Echocardiographic Assessment of the Right Heart in Mice (Video) | JoVE

Echocardiografische Beoordeling van de rechter hart in Muizen

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

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats (Video) | JoVE

Geautomatiseerde Meting van Microcirculatoire snelheid van de bloedstroom in longmetastasen van 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. ...

An Improved Method for Rapid Intubation of the Trachea in Mice

An Improved Method for Rapid Intubation of the Trachea in Mice (Video) | JoVE

Een verbeterde werkwijze voor snelle intubatie van de trachea in muizen

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

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats (Video) | JoVE

Een model van remodellering Door vernauwing van de abdominale aorta bij ratten

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

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals (Video) | JoVE

Wegwerp dosators voor Pulmonary Insufflatie van therapeutische middelen voor kleine dieren

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

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

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System (Video) | JoVE

Het vergelijken van de effecten van elektronische sigarettendamp en sigarettenrook in een roman<em&gt; In Vivo</em&gt; Belichtingssysteem

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

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice (Video) | JoVE

Long fixatie onder constante druk voor de evaluatie van emfyseem bij muizen

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