Name: air-inflation of murine lungs with vascular perfusion-fixation
Uploaded: 2021-02-02T14:00:00
Description: Watch this Scientific Journal Video about Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation at JoVE.com

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>
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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.

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	<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, ...

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