This work was funded by the National Heart, Lung, and Blood Institute (NHLBI) grants HL140039 and HL130938. The authors would like to thank Shannon Hott and Jazalle McClendon for their technical expertise.

All methods described in this protocol have been approved by the Institutional Animal Care and Use Committee (IACUC) of National Jewish Health.
NOTE: The protocol is organized into three components. The first component details the construction of the air-inflation with perfusion/fixation equipment. A second section describes how to set up the equipment for an experiment. The final section describes how to prepare the animal and perform the experiment.
1. Construction ...

<ol>
	<li>Hsia, C. C. W., Hyde, D. M., Ochs, M., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Official+Research+Policy+Statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+Standards+for+Quantitative+Assessment+of+Lung+Structure.">An Official Research Policy Statement of the American Thoracic Society/European Respiratory Society: Standards for Quantitative Assessment of Lung Structure.</a> American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).</li><li>Weibel, E. R., Limacher, W., Bachofen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+rapidly+frozen+lungs:+evaluation+on+the+basis+of+standard+criteria.">Electron microscopy of rapidly frozen lungs: evaluation on the basis of standard criteria.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 53 (2), 516-527 (1982).</li><li>Bachofen, H., Ammann, A., Wangensteen, D., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+fixation+of+lungs+for+structure-function+analysis:+credits+and+limitations.">Perfusion fixation of lungs for structure-function analysis: credits and limitations.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 53 (2), 528-533 (1982).</li><li>Gil, J., Bachofen, H., Gehr, P., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+volume-surface+area+relation+in+air-+and+saline-filled+lungs+fixed+by+vascular+perfusion.">Alveolar volume-surface area relation in air- and saline-filled lungs fixed by vascular perfusion.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 47 (5), 990-1001 (1979).</li><li>Harris, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-Volume+Curves+of+the+Respiratory+System.">Pressure-Volume Curves of the Respiratory System.</a> Respiratory Care. 50 (1), 78-99 (2005).</li><li>Bachofen, H., Sch&#252;rch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+surface+forces+and+lung+architecture.">Alveolar surface forces and lung architecture.</a> Comparative Biochemistry and Physiology Part A: Molecular &amp; Integrative Physiology. 129 (1), 183-193 (2001).</li><li>Mead, J., Takishima, T., Leith, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+distribution+in+lungs:+a+model+of+pulmonary+elasticity.">Stress distribution in lungs: a model of pulmonary elasticity.</a> Journal of Applied Physiology. 28 (5), 596-608 (1970).</li><li>Mariano, C. A., Sattari, S., Maghsoudi-Ganjeh, M., Tartibi, M., Lo, D. D., Eskandari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Mechanical+Strain+Characterization+of+Ventilated+ex+vivo+Porcine+and+Murine+Lung+using+Digital+Image+Correlation.">Novel Mechanical Strain Characterization of Ventilated ex vivo Porcine and Murine Lung using Digital Image Correlation.</a> Frontiers in Physiology. 11, 600492 (2020).</li><li>Braber, S., Verheijden, K. a. T., Henricks, P. a. J., Kraneveld, A. D., Folkerts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema.">A comparison of fixation methods on lung morphology in a murine model of emphysema.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 299 (6), 843-851 (2010).</li><li>Brain, J. D., Gehr, P., Kavet, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+Macrophages.">Airway Macrophages.</a> American Review of Respiratory Disease. 129 (5), 823-826 (1984).</li><li>Wheeldon, E. B., Podolin, P. L., Mirabile, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+Macrophage+Distribution+in+a+Mouse+Model:+The+Importance+of+the+Fixation+Method.">Alveolar Macrophage Distribution in a Mouse Model: The Importance of the Fixation Method.</a> Toxicologic Pathology. 43 (8), 1162-1165 (2015).</li><li>Matulionis, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+deformation+and+macrophage+displacement+in+smoke-exposed+and+normal+mice+(Mus+musculus)+following+different+fixation+procedures.">Lung deformation and macrophage displacement in smoke-exposed and normal mice (Mus musculus) following different fixation procedures.</a> Virchows Archiv. A, Pathological Anatomy and Histopathology. 410 (1), 49-56 (1986).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> JoVE (Journal of Visualized Experiments). (65), e3564 (2012).</li><li>Crosfill, M. L., Widdicombe, 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=Physical+characteristics+of+the+chest+and+lungs+and+the+work+of+breathing+in+different+mammalian+species.">Physical characteristics of the chest and lungs and the work of breathing in different mammalian species.</a> The Journal of Physiology. 158 (1), 1-14 (1961).</li><li>Ramos-Vara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+Methods+of+Immunohistochemistry.">Principles and Methods of Immunohistochemistry.</a> Drug Safety Evaluation: Methods and Protocols. , 115-128 (2017).</li></ol>

Although commonly used, intratracheal-based fixation methods displace leukocytes from the airways and can alter normal lung architecture. The method of air-inflation with vascular perfusion-fixation that is provided in this protocol overcomes these pitfalls and more accurately preserves lung anatomy. The keys to obtaining high-quality tissue from the vascular perfusion-fixation method include careful monitoring of air-inflation pressures, avoidance of air leaks, and ensuring adequate perfusion of fixative into the vascul...

Lung histology represents the gold standard for assessing lung architecture during health and disease and is one of the most commonly used tools by pulmonary researchers1. One of the most critical aspects of this technique is the proper isolation and preservation of lung tissue, since variability in this step can lead to poor tissue quality and erroneous results1,2,3. In living animals, lung volume is determined by the balance between inward elastic recoil of the lung and outward forces transmitted from the chest wall and diaphragm by surface tension. Accordingly, when the thorax is entered, outward forces are lost and the lung collapses. Histologic sections prepared from collapsed lungs have a crowded appearance and boundaries between anatomic compartments (i.e., airspaces, vasculature, and interstitium) can be difficult to distinguish. To circumvent this challenge, researchers often inflate the lungs during chemical fixation so that airspace size and architecture is maintained.
Lungs can be inflated with air or liquid. The pressure necessary to inflate the lungs to the same volume differs between air- and liquid-inflation due to intermolecular forces at the air-liquid interface. Higher pressure (e.g., 25 cmH2O) is required during air-inflation than liquid inflation (e.g., 12 cmH2O) to overcome surface tension and open the collapsed alveoli4. Once alveoli have been recruited, a lower pressure can keep the alveoli open to the same volume as the pressure-volume curve plateaus, and pressures equalize throughout the lung according to Pascal&#39;s law4,5,6,7,8.
Two main methods of lung inflation and fixation exist to preserve murine lungs for histology. Most commonly, the airspaces are instilled with liquid - often containing a fixative. The main advantage of this approach is that it is relatively easy and requires little training. While intratracheal instillation of fixative may be preferred in studies that focus on the vasculature, liquid that is instilled via the trachea tends to push proximal airway cells and mucins into more distal airspace regions while air inflation does not1,3,4,9,10,11. Moreover, inadvertent detachment of leukocytes from the epithelium during liquid inflation alters their morphology, artifactually giving them a simple, rounded appearance4,10,11,12. Finally, inflation of the lungs with liquid can unintentionally compress the interstitium4,10,11. Together, these factors can distort the normal anatomy and cellular distributions within the preserved lungs, thus limiting the technique.
An alternative method of tissue preservation is vascular perfusion-fixation. In this method, fixative is perfused into the pulmonary vasculature via the vena cava or the right ventricle. This method preserves the location and morphology of cells in the airspace lumen. However, unless the lungs are inflated during perfusion-fixation, the lung tissue is likely to collapse.
Air-inflation with vascular perfusion-fixation harnesses strengths from each of the above fixation techniques. Herein we provide a protocol for this technique. The materials and equipment that are required are relatively inexpensive and can be easily obtained and assembled. The completed setup, shown in Figure 1A, provides constant airway pressure to the lungs by way of an adjustable, fluid-filled column while a peristaltic pump delivers fixative via the right ventricle. Lungs with preserved morphology can then be further processed for structure-function analyses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>00117XF-Stopcock 1 way 100/PK M Luer</td>
			<td>Cole-Parmer</td>
			<td>Mfr # VPB1000050N &#8211; Item # EW-00117-XF</td>
			<td>Stopcock</td>
		</tr>
		<tr>
			<td>BD 60 mL syringe, slip tip</td>
			<td>BD</td>
			<td>309654</td>
			<td>Syringe used to construct the water column</td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 25G x 5/8</td>
			<td>BD Biosciences</td>
			<td>305122</td>
			<td>Needle for vascular perfusion/fixation</td>
		</tr>
		<tr>
			<td>Female Luer Thread Style Panel Mount 1/4-28 UNF to Male Luer</td>
			<td>Nordson Medical</td>
			<td>FTLLBMLRL-1</td>
			<td>Female Luer</td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3393</td>
			<td>Heparin solution.</td>
		</tr>
		<tr>
			<td>Luer-Stub Adapter BD Intramedic 20 Gauge</td>
			<td>BD Biosciences</td>
			<td>427564</td>
			<td>Luer-Stub Adapter</td>
		</tr>
		<tr>
			<td>Male Luer (2) to Female Luer Thread Style Tee</td>
			<td>Nordson Medical</td>
			<td>LT787-9</td>
			<td>Male Luer</td>
		</tr>
		<tr>
			<td>Nalgene 180 Clear Plastic PVC Tubing</td>
			<td>ThermoFisher Scientific</td>
			<td>8000-9020</td>
			<td>Tubing</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Aqueous Solution - 32%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>Fixative solution. Diluted to 4% with phosphate buffered saline</td>
		</tr>
		<tr>
			<td>Permatex Ultra Blue Multipurpose RTV Silicone Gasket Maker</td>
			<td>Permatex</td>
			<td>81724</td>
			<td>Silicone Gasket Maker for air-tight sealing of chambers</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline, 1x Without Calcium and Magnesium</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>Bottle used to construct the air-inflation chamber, and buffer used for heparin and fixative solutions</td>
		</tr>
		<tr>
			<td>Sterilite Ultra Seal 16.0 cup rectangle food storage container</td>
			<td>Sterilite</td>
			<td>0342</td>
			<td>Animal processing container</td>
		</tr>
	</tbody>
</table>

air-inflation of murine lungs with vascular perfusion-fixation

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, commonly used instillation-based fixation methods can displace airspace cells and mucus into terminal airways and can alter tissue morphology. In comparison, vascular perfusion-fixation techniques are superior at preserving the location and morphology of cells within airspaces and the mucosal lining. However, if positive airway pressure is not simultaneously applied, regions of the lungs may collapse and capillaries may bulge into the alveolar spaces, leading to distortion of the lung anatomy. Herein, we describe an inexpensive method for air-inflation during vascular perfusion-fixation to preserve the morphology and location of airway and alveolar cells and interstitium in murine lungs for downstream histologic studies. Constant air pressure is delivered to the lungs via the trachea from a sealed, air-filled chamber that maintains pressure via an adjustable liquid column while fixative is perfused through the right ventricle.

In an intact thorax, the lungs are held open by outward forces applied by the chest wall via the pleural space6,14. When the diaphragm is entered during dissection, the integrity of the pleural space is abolished and the lungs should collapse (Figure 2A, 2B). To re-expand the lungs, air inflation is performed. As a first step, 25 cm of water pressure is applied to ensure recruitment of collapsed airspaces. Accordingl...

Watch this Scientific Journal Video about Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation at JoVE.com

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Presented is a method for air-inflation with vascular perfusion-fixation of the lungs that preserves the location of cells within airways, alveoli and interstitium for structure-function analyses. Constant airway pressure is maintained with an air-inflation chamber while fixative is perfused via the right ventricle. Lungs are processed for histologic studies.

Lung histology is often used to investigate the contributions provided by airspace cells during lung homeostasis and disease pathogenesis. However, ...

This is an inexpensive method that combines the advantages of air inflation and vascular perfusion fixation for preserving lungs for structural and functional analyses. The main advantage of this technique is the preservation of both cell morphology and location within the airspaces of the lung. Care should be taken while placing the lure stub adapter into the trachea and the perfusion needle into the right ventricle to ensure adequate inflation and fixation.To set up the air-inflation apparatus, place a syringe for the water column into a ring holder and measure a vertical height of 25 centimeters from the animal platform to 25 centimeters on the water column. Attach to the end of the water column tube to the stopcock on the air chamber and attach a tube from the female lure of the air chamber to the stopcock on the animal processing container. Confirm that the cap to the air chamber and the stopcock on the outside of the animal processing container are closed, and that the stopcock on the tubing leading from the water column to the air inflation chamber is open.Then fill the syringe with water to the 25 centimeter mark. Water will flow through the syringe and tubing into the air chamber. Once the pressure is equalized, the water will stop flowing.To prepare the lungs for inflation make one lateral incision in the abdominal wall below the rib cage of the euthanized mouse, and a second lateral abdominal wall incision above the hips. Cut along the midline from the interior incision toward the superior incision, and to use blunt scissors to carefully make an incision into the lateral side of the diaphragm. The lungs should collapse as soon as the diaphragm is punctured.Cut transversely along the diaphragm to open the thoracic cavity and cut superiorly along the sternum from the xiphoid process to the juggler notch, and laterally above the rib cage to fully expose the heart and lungs. Pin down the sides of the rib cage and make a midline incision in the neck, above the trachea. Remove the skin, muscle, thyroid gland, and connective tissue surrounding the trachea and use curved forceps to slide two pieces of suture under the posterior trachea.Use one piece of suture to hold the inflation lure stub adapter in place and use an 18 gauge by one inch needle to make a small hole in the trachea. Insert a 20 gauge lure stub adapter into the hole and tie the suture around the trachea immediately distal to where the lure stub adopter enters the hole. Then transfer the animal to the animal processing container and attach the lure stub adapter to the female lure on the inside of the animal processing container.To inflate the lungs, place the 25 gauge by 5/8th inch needle attached to the perfusion apparatus tubing into the right ventricle of the heart and cut the abdominal aorta to allow blood to drain from the heart and to promote the flow of perfusate through the lungs. Open the stopcock on the outside of the animal processing container and inflate the lungs at 25 centimeters of water for five minutes, while taking care that the water level in the syringe does not decrease too quickly. During the last minute of the lung inflation, turn on the peristaltic pump to a flow rate of 10 milliliters per minute.Heparin solution should flow from the bottle through the tubing into the animal. After inflating the lungs for five minutes, turn off the pump and switch the perfusate from heparin to fixative. Lower the water column syringe to 20 centimeters.It's normal for air bubbles to move within the water column as the pressure changes. Check the water level in the syringe. It should be at the 25 centimeter mark.Then wait one minute to allow the lungs to deflate from 25 to 20 centimeters of water pressure before restarting the pump at a 6.5 milliliter per minute flow rate for 10 to 15 minutes, vascular fixative perfusion. For extraction of the inflated and fixed lung tissue, tightly tie the second piece of thread around the trachea, distal to the lure stub adapter, and remove the lure stub adapter from the trachea. Remove the needle from the heart and use blunt scissors to cut the connective tissue, posterior to the mediastinum, to free the lungs and heart from the thoracic cavity taking care to avoid puncturing the lungs.Carefully remove the heart from the lungs and place the lungs into 20 to 25 milliliters of fixative in a 50 milliliter conical tube. Pull the suture holding the trachea through the opening in the conical tube and secure the suture by the threads of the cap. Then invert the tube to ensure that the buoyant, air-inflated lungs remain fully submerged in fixative and process the lungs for histologic studies according to standard protocols.When the diaphragm is entered during dissection, the integrity of the pleural space will be abolished and the lungs should collapse. Upon the application of 25 centimeters of water pressure, air will enter the lungs via the trachea and inflation should be easily observed. Once the lungs have fully expanded, the inflation pressure can be decreased to 20 centimeters to keep the lungs inflated without overdistension.The lungs should remain inflated after tracheal ligation and removal from the thorax. Deflation can occur if the lungs are punctured during the animal preparation or lung extraction. Histological analysis of inflated lung sections reveals that very few immune cells are present in the airway lumen of tissues fixed using traditional liquid-based inflation.In contrast, inflammatory cells are preserved within the airspaces of tissues fixed via vascular perfusion with air inflation. Following air inflation with profusion fixation, lung tissue may be embedded in formalin for tissue sectioning and subsequent analysis via histology techniques, including staining, immunohistochemistry, and immunofluorescent imaging.

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6.3K Views.  National Jewish Health. This is an inexpensive method that combines the advantages of air inflation and vascular perfusion fixation for preserving lungs for structural and functional analyses. The main advantage of this technique is the preservation of both cell morphology and location within the airspaces of the lung. Care should be taken while placing the lure stub adapter into the trachea and the perfusion needle into the right ventricle to ensure adequate inflation and fixation.To set up the air-inflation...