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, 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.
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'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.
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 of the water column apparatus (Figure 1B)
2. Construction of the air-inflation chamber (Figure 1C)
3. Construction of the animal-processing container (Figure 1D)
4. Preparation of solutions
5. Preparation of perfusion apparatus
NOTE: A peristaltic pump is suggested for delivery of fluids into vasculature to ensure constant flow rate. The following directions are for setting up the peristaltic pump and may be different for each model. Alternatively, if a peristaltic pump is unavailable, a second water column apparatus may be constructed to perfuse fluids from a height of 35 cm H2O.
6. Preparation of air-inflation apparatus
7. Preparation of animals (Figure 2)
NOTE: This procedure has been modified from Gage et al13. We have completed this procedure on adult male and female mice of varying ages and note no age or sex bias.
8. Air inflation, perfusion and fixation of the lungs (Figure 2)
9. Extraction of lungs (Figure 3)
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. Accordingly, when the stopcock outside the animal holding container is opened, air will enter the lungs via the trachea and inflation should be easily observed (Figure 2C). Once the lungs are fully expanded, the inflation pressure is decreased to 20 cm water pressure (Figure 2D). The 20 cm of water pressure is chosen because it maintains complete inflation of the lungs but does not over-distend the airspaces.
The lungs should remain inflated after tracheal ligation (Figure 3A) and after removal from the thorax (Figure 3B). Deflation of lungs (Figure 3C) can occur if lungs are punctured during animal preparation or extraction. Adding fixative to the pleural surface may help seal minor leaks during the procedure; however, fixative should be applied cautiously as excess may adhere lungs to the thoracic cavity. Any leaks that are not sealed during fixation will result in collapsed lungs upon removal from the air-inflation apparatuses. Deflation of the lungs may also occur if the trachea is not completely tied off. When submerged in fixative, properly inflated lungs will have greater buoyancy than deflated lungs.
Inflated lungs can then be processed for histological analyses according to established protocols1,15. For Figure 4, lungs were processed for frozen sectioning and stained with a commercial manual staining system. Very few immune cells are present in the airway lumens of tissue fixed using traditional liquid-based inflation (Figure 4A). In contrast, inflammatory cells are preserved throughout airspaces in tissue fixed via vascular perfusion with air-inflation (Figure 4B).
Figure 1: Assembly of apparatuses. A. Full assembly of all apparatuses. B. The water column consists of a 60 mL syringe connected to the air-inflation chamber via 180 PVC tubing and a two-way male Luer. C. A 500 mL sealed plastic container was used to construct the air-inflation chamber. The water column's male Luer connects to a stopcock connected to a female Luer within the walls of the chamber. An additional female Luer connects tubing from the air-inflation chamber to the animal processing container. Both female Luers are coated in silicone gasket maker to ensure an air-tight seal. Two male Luers are connected to both ends of tubing which connects the air-inflation chamber to the animal-processing container. D. Animals are attached to the air-inflation chamber through a 20G Luer stub adapter placed through a hole into the trachea. The Luer stub adapter is connected to a female Luer within the walls of the animal processing container. Please click here to view a larger version of this figure.
Figure 2: Animal preparation, connection to the air-inflation chamber, and inflation of lungs. A. After euthanasia, the animal's peritoneal and thoracic cavities are exposed. The rib cage is removed or pinned down to allow expansion of the lungs upon inflation. A Luer stub adapter is inserted into a small hole cut into the trachea and secured with thread or suture. The Luer stub adapter is connected to a female Luer within the wall of the animal processing chamber. The other end of the female Luer is attached to a stopcock to control air flow from the air-inflation chamber (not shown). B. Collapsed lungs before air inflation. C. Lungs are inflated to 25 cm water pressure to recruit atelectasic regions. D. When the pressure is changed to the intended fixation pressure (20 cm water), the lungs deflate slightly. Also depicted is placement of a 25G x 5/8 needle into the right ventricle for vascular perfusion-fixation of the lung. All images are photographs at 15.9 megapixel resolution and at 4:3 aspect ratio. Please click here to view a larger version of this figure.
Figure 3: Extraction of lungs following completion of air-inflation with vascular perfusion fixation. A. The trachea is tied off distal to the Luer stub adapter and lungs are extracted by cutting the connective tissue posterior to the mediastinum. B. Air-inflated lungs after successful procedural completion. C. Example of poorly inflated lungs that resulted from an air leak that occurred within the air-inflation chamber. Note these lungs are smaller than the successfully inflated lungs. All images are photographs at 15.9 megapixel resolution and at 4:3 aspect ratio. Please click here to view a larger version of this figure.
Figure 4: Comparison of lung tissue obtained by intratracheal-based fixation versus air-inflation with vascular perfusion-fixation. A. Lungs preserved by intratracheal-based fixation. B. Lungs preserved by air-inflation with vascular perfusion-fixation. B (inset). Arrows show leukocytes in the airways of a lung fixed by vascular perfusion-fixation; stars highlight leukocytes in the alveoli. In comparison, leukocytes are notably absent in the airways of lungs fixed via the intratracheal route (A inset) and intra-alveolar leukocytes are displaced, appearing to be in tight contact with epithelial cells. Abbreviations: A– airway, V– vessel. Magnification of images are 40x with 100x and 200x for A and B insets respectively. Please click here to view a larger version of this figure.
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 vasculature.
One limitation to this procedure is that when the integrity of the thorax is interrupted, the lungs collapse and re-inflation of the lungs after collapse is necessary for accurate histological assessment. An alternative to the protocol that would maintain lung inflation without collapse would be the use of a small animal ventilator. However, such equipment is often expensive, and the protocol here offers an inexpensive solution. In healthy lungs, surfactant produced by alveolar epithelial cells helps to reduce surface tension, and in most cases lungs can be easily re-expanded. However, in diseased lungs, tissues can be stiffer and lung surfactant function can be altered, promoting lung collapse. To mitigate this effect, collapsed areas can be “recruited” using slightly higher air inflation pressures (i.e., 25 cmH2O)5. The pressure can then be reduced to allow slight deflation of the lungs to physiologic size. In our hands, an inflation pressure of 20 cm water works well. Pressures higher than this can over-distend the alveoli and impair vascular perfusion. Conversely, low pressures result in airspace collapse. Along similar lines, vascular perfusion pressures must also be titrated. Excessive perfusion pressures may distend capillaries into the alveolar space or even damage the capillaries and cause lung edema4. On the other hand, if vascular perfusion pressures are too low, perfusion may be inadequate. We have found that flow rates of 10 mL/min for the heparin solution and 6.5 mL/min for the fixative solution achieve an optimal result.
Checking the air inflation chamber for leaks is imperative to ensure constant inflation pressure during vascular perfusion-fixation. Once water is added to the syringe it should flow into the bottom of the air inflation chamber until pressures equalize. A small amount of additional water may need to be added to the syringe to maintain a column height of 25 cm for inflation and 20 cm for fixation. Silicone sealant may need to be replaced if flow into the air inflation chamber does not cease.
Another cause of air leakage is damage to the lungs. This most commonly occurs during opening of the thoracic cavity or during extraction of the lungs from the thorax. Thus, practice and great care must be taken to avoid damaging the lung during mouse preparation. A less common cause is lung pathology that results from severe lung disease. Clues to air leaks from the lungs include slow emptying from the fluid column in the syringe, a hissing sound or bubbles coming from the lung surface. Applying a small amount of fixative to the lungs at the site of the leak can help seal small leaks; however certain fixative can cause adherence of the lungs to the thoracic cavity and when lung damage is extensive, the lungs may still collapse once air pressure is removed.
Once any sources of air leakage have been assessed and managed, lungs should inflate and remain inflated during fixation. The trachea should be ligated below the cannula before removal from the inflation apparatus to prevent collapse. Lungs can then be processed for histologic studies. Air-inflation with vascular perfusion-fixation of lungs aims to preserve numbers, morphology and location of airway cells while adequately preserving global lung architecture for histologic structure-function studies.
The authors have nothing to disclose.
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.
00117XF-Stopcock 1 way 100/PK M Luer | Cole-Parmer | Mfr # VPB1000050N – Item # EW-00117-XF | Stopcock |
BD 60 mL syringe, slip tip | BD | 309654 | Syringe used to construct the water column |
BD PrecisionGlide Needle 25G x 5/8 | BD Biosciences | 305122 | Needle for vascular perfusion/fixation |
Female Luer Thread Style Panel Mount 1/4-28 UNF to Male Luer | Nordson Medical | FTLLBMLRL-1 | Female Luer |
Heparin sodium salt from porcine intestinal mucosa | Sigma-Aldrich | H3393 | Heparin solution. |
Luer-Stub Adapter BD Intramedic 20 Gauge | BD Biosciences | 427564 | Luer-Stub Adapter |
Male Luer (2) to Female Luer Thread Style Tee | Nordson Medical | LT787-9 | Male Luer |
Nalgene 180 Clear Plastic PVC Tubing | ThermoFisher Scientific | 8000-9020 | Tubing |
Paraformaldehyde Aqueous Solution – 32% | Electron Microscopy Sciences | 15714-S | Fixative solution. Diluted to 4% with phosphate buffered saline |
Permatex Ultra Blue Multipurpose RTV Silicone Gasket Maker | Permatex | 81724 | Silicone Gasket Maker for air-tight sealing of chambers |
Phosphate-Buffered Saline, 1x Without Calcium and Magnesium | Corning | 21-040-CV | Bottle used to construct the air-inflation chamber, and buffer used for heparin and fixative solutions |
Sterilite Ultra Seal 16.0 cup rectangle food storage container | Sterilite | 0342 | Animal processing container |