We wish to thank Francesca Polverino MD, a Research Fellow at Brigham and Women&#8217;s Hospital for her contribution to this article, and also Monica Yao, BS, and Kate Rydell, BS for their assistance with murine husbandry and exposing the mice to cigarette smoke.&#160;&#160;
This work was supported by Public Health Service, National Heart, Lung, and Blood Institute Grants HL111835, HL105339, HL114501, Flight Attendants Medical Research Institute Grant #CIA123046, the Brigham and Women&#8217;s Hospital-Lovelace Respiratory Research Institute Consortium, and the Cambridge NIHR Biomedical Research Centre.

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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...

The use of animal models to study COPD is challenging because no model can perfectly replicate all features of the human disease(2). Most investigators use mice to model COPD because of the similarities between mice and humans in their pulmonary physiology, pathology, genetics, and metabolites. Also, mice are relatively inexpensive to study, and both emphysema and small airway remodeling develop within 6 months of CS exposure(5,7-9).
Cigarette smoke-induced COPD: Several methods can induce COPD in mice. Most researchers expose mice to CS, which is the main etiologic factor for human COPD. CS exposure for 6 months causes the development of emphysema and small airway remodeling (SAR) in mice, but the severity of the disease that is induced varies depending on the murine strain studied. For example, NZWLacZ mice are resistant to the development of CS-induced emphysema whereas AKR/J mice are extremely sensitive(10). Most investigators study C57BL/6 strain mice in the CS exposure model as many gene-targeted mice are available in this strain. After 6 months of CS exposure, emphysema and small airway fibrosis develop in wild type (WT) C57BL/6 mice, and both lesions are relatively mild in severity(5,10). Researchers use two types of CS exposure: nose-only and whole-body exposures. The major disadvantages of the nose-only exposure technique are that: 1) it is a more labor-intensive method; and 2) mice have to be restrained in small chambers which can induce a stress response and hyperthermia in the animals(11). The major disadvantage of whole-body exposure (described herein) is that the animals can ingest (as well as inhale) nicotine and tar products when they clean their fur. Mice exposed to whole-body CS also have lower carboxyhemoglobin levels and reduced loss of body weight when compared with animal exposed to nose-only CS(12).
Pulmonary function test (PFTs): Measures of lung compliance and elastance are usually similar in C57BL/6 wild type (WT) mice exposed to air or CS for 6 months due to the relatively mild emphysema that develops when this strain is exposed to CS(10). However, when emphysematous destruction is more severe, increases in lung compliance and left shifts in the pressure-volume (PV) flow loops can be detected. The latter can be observed, for example, in murine strains that are more susceptible to the effects of CS(10), in CS-exposed C57BL/6 strain gene-targeted mice that have a more severe emphysema type than C57BL/6 WT mice(13), or in CS-exposed mice subjected to environmental changes that render them more susceptible to the effects of CS(14). This protocol uses a small animal ventilator to measure reductions in the elastic recoil of the lung (increases in quasistatic lung compliance [Cst] and reductions in tissue elastance [H]), PV flow loops, and changes in airway and tissue resistance in anesthetized mice(15,16).
Measures of pulmonary emphysema: Analysis of emphysema development in CS-exposed C56BL/6 strain mice is challenging because its distribution is spatially heterogeneous. Several different methods quantify airspace enlargement in mice. The first method used was the mean linear intercept (Lm)(17). However, the Lm method is a slow, manual process which may not capture the heterogeneity of the disease (unless all sections of the lung are randomly sampled) and its use may therefore introduce observer bias into the analysis. The destructive index [DI,(18)] also quantifies airspace enlargement using a transparent sheet with 50 equally distributed points placed over a printed digitized image of a hematoxylin and eosin-stained lung section. The PI method scores the area surrounding each point according to the extent to which the alveolar ducts and alveolar walls within this area are destroyed. The main disadvantage of the DI method is that it is time-consuming and not more accurate than other methods(19,20).
This protocol measures mean alveolar chord length and alveolar area on paraffin-embedded lung sections stained with Gill&#8217;s stain. Morphometry software converts images of lung sections to binary images (in which tissue is white and airspace is black), and then superimposes a uniform grid of horizontal and vertical lines (chords) and the software then quantifies the length of each chord within areas identified by software as airspace. Using this method, it is possible to measure the size of the alveoli in all parts of the lung in a standardized and relatively automated manner(21).
Small airway remodeling (SAR): The increased deposition of ECM proteins (especially interstitial collagens) around small airways occurs in CS-exposed animals and contributes to airflow obstruction. Researchers do not study SAR in animal models of COPD as frequently as emphysema development(22). To quantify SAR in CS-exposed mice, this protocol uses image analysis software to measure the thickness of the layer of ECM proteins that is deposited around the small airways (airways having a mean diameter between 300 and 899 m) in paraffin-embedded lung sections stained with Masson&#8217;s trichrome stain.

<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.

This protocol begins with whole-body exposure of mice for CS. Adequate oversight and maintenance of the device and monitoring of TPM counts ensure consistent smoke exposures (Figure 1). It is important that the researcher practices the lung inflation technique using the inflation device
This protocol begins with whole-body exposure of mice for CS. Adequate oversight and maintenance of the device and monitoring of TPM counts ensure consistent smoke exposures (Figure 1</...

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.

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

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13.2K vistas.  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. 

Video: 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.

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

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.

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.

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.

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.

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.

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.

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.

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

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

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.

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