This work was supported in part by JSPS KAKENHI Grant Number 26461199 (T. Sato) and the Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Grant Number E2920 (T. Sato). The funder had no role in the design of the current methods and in writing the manuscript.

The following methods have been approved by the Animal Care and Use Committees of Juntendo University School of Medicine. The Guidelines for Proper Conduct of Animal Experiments, Science Council of Japan, June 1, 2006 were followed. There are three main steps in this method: 1) mouse dissection, 2) lung exsanguination, and 3) fixation of lung tissues assisted by specialized equipment. Typically, lung specimens are processed to embedment after 48 h of fixation12,15,16,17,18.
1. Mouse dissection
<ol>
	<li>Measure the body weight of the mouse, then determine the amount of pentobarbital to administer.</li>
	<li>Inject pentobarbital intraperitoneally at a dosage of 70 mg/kg of body weight and confirm anesthesia by the absence of reaction to toe pinch.</li>
	<li>Inject the needle at a 45&#176; angle until it penetrates the skin and muscle. Draw the plunger and confirm an air vacuum, then inject the pentobarbital.</li>
	<li>Confirm anesthesia by the absence of reflex motion. 
	NOTE:&#160;Using anesthetized mouse as opposed to a euthanized mouse is recommended for fully lung exsanguination.</li>
	<li>Cut the mouse skin and abdominal muscle at the medial line, aiming for the cephalic area.</li>
	<li>Cut laterally to provide a wider working space.</li>
</ol>
2. Lung exsanguination
<ol>
	<li>Expose the diaphragm layer and puncture it with forceps.</li>
	<li>Open the thoracic space and cut the sternal area, allowing the lungs and heart to be seen clearly.</li>
	<li>Cut the heart in left atrium and right ventricle.</li>
	<li>Insert a cannula (24&#160;G) into the right ventricle area and direct it to the cephalic area until it reaches the pulmonary artery, as shown in Figure 1.</li>
	<li>Turn on the pump and allow the 1x phosphate-buffered saline (PBS) to circulate (approximately 200 mL/h) until all lung tissue changes to a white color.</li>
</ol>
3. Fixation of lung tissue
<ol>
	<li>Remove the trachea, lungs, and heart.</li>
	<li>Free all three organs by cutting the surrounding connective tissues.</li>
	<li>Tie the right main bronchus with a suture thread and cut all lobes of the right lung.</li>
	<li>(Optional): the lobes of the right lung consist of four parts. Cut these parts from the right main bronchus and divide the parts for processing as frozen tissue samples.</li>
	<li>Insert the heart and the lobes of the left lung into fixing agents, located inside a 10 mL syringe. 
	CAUTION: Fixing agents are hazardous. Wear proper protective equipment (e.g., long rubber gloves) and work in a well-ventilated room.</li>
	<li>Create a vacuum condition using a 10 mL syringe to inflate the lung, as shown in Figure 2.</li>
	<li>Insert a cannula (20 G) into the trachea and tie a knot.</li>
	<li>Inflate the lung with fixing agents to check with for leaks, using a 1 mL syringe.</li>
	<li>Transfer to lung fixation pressure equipment, as illustrated in Figure 3.</li>
	<li>After fixing periods, remove the lung sample tying the trachea off with a knot.</li>
</ol>

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The authors have no competing interests to declare.

The fixation procedure for rodent lungs presented here is not novel; however, this system has several advantages. Firstly, it can fix many lungs (maximum of 20) with the same condition at one time. The Society of Toxicologic Pathology states that the pressure for gravity instillation vary from 22&#8211;25 cmH2O22. Notably, several studies have performed lung fixation at a pressure of 25 cmH2O13,19,23,24,25,26,27, which has been adopted in our laboratory using the current system12,15,16,17,18.
Secondly, it can fix lung tissues at a constant pressure for various periods of time. In our laboratory, lung samples are usually fixed for 48 h. Many investigators use a relatively short period of time (e.g., 5&#8211;20 min)13,28,29,30,31,32, then tie off the inflated lung and immerse in formalin for extended periods as desired. There is no data or research indicating a gold standard for the length of duration for lung fixation. However, the statement of the American Thoracic Society (ATS)/European Respiratory Society (ERS) describe the &#8220;silver standard&#8221;, in which airway inflation pressure must be maintained for at least 24 h14. The Japanese Society of Pathology also recommended fixation times of no longer than 1 week to produce consistent immunohistochemical slides; although, their recommendation is based on analysis using human specimens33. Relatively short fixing time periods may not be applicable to the current system, because each sample is supposed to be individually placed in the lower container. This is a limitation of the current system. In conclusion, the proper length of time for mouse lung fixation remains unknown.
Critical steps in this method are related to the risk of lung formalin leakage during the formalin fixation process. Lung formalin leakage can cause lung size shrinkage. This risk can be divided into two parts. The first part occurs during the sacrifice step. While opening the thoracic cage, it is important not to cause injury to the lung surface. The key to prevention is to approach this from the diaphragm and continue to cut the thoracic rib cage after the lung is detached from the parietal pleura. This method avoids lung injury caused by surgical equipment. Another key step occurs when tying the right main bronchus. It is important to identify which are the mouse&#8217;s right lobes. Placing the lungs in a position where they can be seen from a dorsal view enables easier identification of the location of the lungs.
The second part is during the lung fixation process using specialized equipment. A critical step occurs while inserting the lung specimen into the lower container&#8217;s formalin port. It should be confirmed that the insertion is tightly secured to prevent lung specimen detachment from the formalin port during the constant pressurization process. Another aspect to highlight is the tubing connection between the three parts of specialized equipment (lower container, upper container, and pump). All tube connections should be tightly connected. If leakage occurs, the formalin volume in the upper container will decrease, thereby reducing constant pressure.
According to recommendations from the Society of Toxicologic Pathology, intratracheal instillation of formalin has advantages for rodent lung model, which prevail over its disadvantages22. They have suggested the use of an intratracheal formalin fixation method when performing quantitative studies of alveolar lung morphometry. Intratracheal lung instillation has two advantages, including preservation of the airway and alveolar wall as well as visualization of lung parenchyma22. One study by Braber et al. revealed that the intratracheal formalin instillation method is superior in terms of preserving lung structure when compared with the vacuum inflation and whole-body perfusion methods13. The current method utilizes intratracheal instillation in a mouse model to optimize visualization of the alveolar area.
Regarding fixing agents, 10% formalin, which contains formaldehyde, is conventionally used. Formaldehyde is widely used as a fixing agent for immunopathological investigations because it does not completely destroy protein immunogenicity. However, the ATS/ERS statement does not recommend formalin fixation, because it does not adequately stabilize tissue structure14. Glutaraldehyde is recommended for airway instillation instead; however, it is subject to destroy protein immunogenicity, which results in an unsuitable fixing agent for immunohistochemistry. Several pieces of evidence have reported that the fixed lungs may be provided for morphometric evaluation (e.g., mean linear intercepts, internal surface area, and destructive index) following formalin fixation using the current fixation system12,15,16,17,18. Certainly, glutaraldehyde can be adopted for the current system; thus, researchers can choose both agents in the current system according to experimental needs.

COPD is one of the leading worldwide causes of death1. Cigarette smoke is the most important cause of COPD, but the mechanisms of pathogenesis remain incompletely defined. COPD demonstrates two main characteristics, including progressive limitation of airflow and an abnormal inflammatory response of the lung. Emphysematous disorder frequently occurs in the lungs of COPD patients2. The pathological findings of emphysema are characterized by alveolar wall destruction3. Several animal species have been used to generate COPD models in vivo (i.e., dogs, guinea pigs, monkeys, and rodents)4. However, the mouse has become the most commonly used in the construction of COPD models. This has many advantages, including its low cost, ability to be genetically modified, extensive genomic information availability, availability of antibodies, and ability to use a variety of mouse strains5. Presently, there is no mouse model that can mimic the full features of human COPD; thus, individual researchers must choose which model is most suitable for the specific COPD research6. The emphysematous mouse model is one of many COPD mouse models that are currently available. Additional models include the exacerbation mouse model, systemic co-morbidities model, and COPD susceptibility model7.
The emphysematous mouse model can be generated by several types of exogenous agents, including chemical agents and cigarette smoke exposure4. Chemical exposure (e.g., to elastase) produces a severe type of emphysema, while cigarette smoke results in mild emphysema8,9. Cigarette smoke is believed to be the main cause for the pathogenesis of COPD; therefore, the choice of cigarette smoke as a means to create a COPD mouse model is reasonable10. Many studies have used cigarette smoke to create emphysema in the mouse. For example, Nikula et al. successfully created an emphysematous mouse model from B6C3F1 female mice by exposing them to cigarette smoke for 7 or 13 months11. We have also established an emphysematous mouse model via senescence marker protein/SMP-30 KO mice12. It is crucial to perform a lung fixation method that can properly visualize this mild emphysema model by cigarette smoke exposure.
Various methods for lung fixation have been established13. However, there is no gold standard method of lung tissue fixation for evaluating emphysema14. Several studies from this lab have shown that the fixation system presented here is useful by creating a stable condition for evaluating emphysema12,15,16,17,18. The main advantage of the current system is that it can fix many lungs with the same condition at one time without lung collapse or deflation. The current lung fixation system uses some special equipment that allows lung specimens to be inflated at an appropriate constant pressure for a given period. This special equipment consists of three parts, including a lower container, upper container, and pump. Lung specimens are placed in the lower container that is connected to pressurized fixing agents, resulting in a 25 cmH2O pressure difference in the level of agents between the upper and lower containers19.

<table><tbody><tr><td>10% formalin (formalin neutral buffer solution)</td><td>Wako</td><td>060-01667</td><td></td></tr><tr><td>Bent forceps</td><td>Hammacher</td><td>HSC187-11</td><td></td></tr><tr><td>Cannula, size 20G</td><td>Terumo</td><td>SR-FS2032</td><td></td></tr><tr><td>Cannula, size 22G</td><td>Terumo</td><td>SR-OT2225C</td><td>Cannula to exsanguinate lung</td></tr><tr><td>Forceps</td><td>Hammacher</td><td>HSC184-10</td><td></td></tr><tr><td>Kimtowel</td><td>Nippon Paper Crecia (Kimberly Clark)</td><td>61000</td><td></td></tr><tr><td>Kimwipe</td><td>Nippon Paper Crecia (Kimberly Clark)</td><td>62011</td><td></td></tr><tr><td>Lower container (acrylic glass material)</td><td>Tokyo Science</td><td>Custom-made</td><td>Pressure equipment component</td></tr><tr><td>Roller pump</td><td>Nissin Scientific Corp</td><td>NRP-75</td><td>Pump machine to exsanguinate lung</td></tr><tr><td>Roller pump RP-2000</td><td>Eyela (Tokyo Rikakikai Co. Ltd)</td><td>160200</td><td>Pressure equipment pump</td></tr><tr><td>Silicone tube &amp;Oslash; 9 mm</td><td>Sansyo</td><td>94-0479</td><td>Pressure equipment component</td></tr><tr><td>Somnopentyl (64.8 mg/mL)</td><td>Kyoritsu Seiyaku</td><td>SOM02-YA1312</td><td>Pentobarbital Sodium</td></tr><tr><td>Surgical scissor</td><td>Hammacher</td><td>HSB014-11</td><td></td></tr><tr><td>Suture thread, size 0</td><td>Nescosuture</td><td>GA01SW</td><td></td></tr><tr><td>Syringe, 1 mL</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Syringe, 1 ml with needle</td><td>Terumo</td><td>SS-01T2613S</td><td></td></tr><tr><td>Syringe, 10 mL</td><td>Terumo</td><td>SS-10ESZ</td><td></td></tr><tr><td>Three-way stopcock</td><td>Terumo</td><td>TS-TR1K01</td><td></td></tr><tr><td>Upper container (acrylic glass material)</td><td>Tokyo Science</td><td>Custom-made</td><td>Pressure equipment component</td></tr></tbody></table>

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.

As described previously, the specialized equipment, which generates extended constant pressure, can be divided into three parts (Figure 3A). The lower part is the point at which to insert the lung sample (Figure 4A). The lung is connected via a cannula (20 G) to the tip of formalin flow using a three-way stop cock (Figure 4B). Pressure is generated from the different surface levels of fixing agents between the lower and upper containers (Figure 5). The pressure difference is 25 cmH2O; however, using the height adjustment knob, the pressure can be adjusted within the range of 25&#8211;30 cmH2O (Figure 5). A pump connects the lower and upper containers via tubes (Figure 3A), preserving a 25 cm difference in fixing agent surface height. The direction of agent flow is described in Figure 3B.
Presented next is a representative result of histological findings in the lung, following 48 h of fixation. Six-month-old male SMP30-KO mice were exposed to cigarette smoke or fresh air (as control) for 8 weeks. Both tissue specimens were stained with hematoxylin and eosin. Figure 6A shows histological findings from the air-exposed mice, which did not exhibit marked airspace enlargement. In contrast, Figure 6B reveals significant airspace enlargement and alveolar wall destruction in mice that were exposed to chronic cigarette smoke.
The mean linear intercepts (MLI) was determined according to the method described by Thurlbeck et al.20 to access the airspace size. The destructive index (DI) was determined to evaluate the destruction of the alveolar wall according to the method described by Saetta et al.21. These morphometric examinations of the lung specimen revealed that DI and MLI were significantly greater in the smoke-exposed SMP30-KO mice than in the air-exposed mice (Figure 6C,D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58197/58197fig1.jpg" /> 
Figure 1: Lung exsanguination. A cannula was inserted at the location of the right ventricle and directed to the pulmonary artery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58197/58197fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58197/58197fig2.jpg" /> 
Figure 2: Vacuum syringe lung inflation. Vacuum condition inside the 10 mL syringe containing fixing agents to inflate the lungs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58197/58197fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58197/58197fig3.jpg" /> 
Figure 3: Lung fixation equipment. (A) The acrylic equipment allowed a 25 cmH2O pressure difference to inflate the lungs continuously for 48 h, utilizing a pump machine. (B) The direction of fixing agent flow is indicated by arrows. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58197/58197fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58197/58197fig4.jpg" /> 
Figure 4: Lower container. (A) The mouse lung specimen was positioned inside fixing agents in the lower container. (B) Inside the lower container, there is a sample placement box, at the top of which formalin flows through a three-way stopcock and the cannula. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58197/58197fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58197/58197fig5.jpg" /> 
Figure 5: Upper container and height adjustment knob. The upper container generated a pressure of 25 cmH2O. There are two pairs of height adjustment knobs which can be used to adjust the height of the upper container; as a result, the pressure that is generated can be set within the range of 25&#8211;30 cmH2O. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58197/58197fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58197/58197fig6.jpg" /> 
Figure 6: Mouse lung histologic and morphometric findings. Representative histologic images of lung sections from 8-week cigarette smoke-exposed or air-exposed SMP30-KO mice (6 month-old, male), stained with hematoxylin-eosin. Scale bar = 100 &#956;m. (A) The air-exposed group did not show significant enlargement or other findings. (B) The cigarette smoke-exposed group showed marked airspace enlargement and alveolar wall destruction. (C) The mean linear intercepts (MLI). In the lungs of cigarette smoke-exposed mice, MLI was significantly greater than air-exposed mice (*p &#60; 0.001). (D) The destructive index (DI). In the lungs of cigarette smoke-exposed mice, DI was significantly increased compared to the lungs of air-exposed mice (*p &#60; 0.001). Values are presented as mean &#177; SD (n = 6 for each group). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58197/58197fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice at JoVE.com

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

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 ...

lung-fixation-under-constant-pressure-for-evaluation-emphysema

Nanyang Technological University

Research

JoVE Journal

Medicine

8.9K Views.  Juntendo University. De constante druk in het fixatieapparaat behoudt de longen in een redelijk opgeblazen toestand, waardoor een histologisch longexemplaar ontstaat dat geschikt is voor de evaluatie van milde sigarettenrook geïnduceerde emfyseem. Het belangrijkste voordeel van dit model is dat het veel longen tegelijkertijd kan repareren met dezelfde constante druk zonder instorting of deflatie van de longen. Het aantonen van de procedure zal Naoko Arano een afgestudeerde student van mijn laboratorium.Na...

Video: Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Perfusion and Inflation of the Mouse Lung for Tumor Histology

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform necropsies rapidly and efficiently from studies without sacrificing the quality of the tissues procured. The goal of this protocol is to present a method to rapidly perfuse, inflate, and fix mouse lungs for downstream histological analysis. This method does not standardize lung inflation; thus, it does not require any special procedures or equipment and instead simply instills fixative directly through the trachea following perfusion through the heart. This allows for sufficient estimation of tumor size, histology, and scoring. This also allows for the collection of frozen tissue prior to lung tissue fixation. This method is limited in that it does not allow for later morphometric quantification of the lung; however, it is more than sufficient for lung tumor analysis from genetically engineered mouse models (GEMMs), syngeneic models, as well as xenograft tumor and metastasis studies.

The purpose of this method is to present a simple and efficient method for the perfusion, inflation, and fixation of mouse lungs for the examination of lung tumor pathology and evaluation of metastases to the lung.

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

Cancer Research

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.

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

Developmental Biology

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

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.

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 ...

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.

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

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

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

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

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

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Biology

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.

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).

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

Lung Fixation under Constant Pressure for Evaluation of Emphysema in Mice

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