Published: February 2nd, 2021
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. ....
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 .......
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.......
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.......
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
|Mfr # VPB1000050N – Item # EW-00117-XF
|BD 60 mL syringe, slip tip
|Syringe used to construct the water column
|BD PrecisionGlide Needle 25G x 5/8
|Needle for vascular perfusion/fixation
|Female Luer Thread Style Panel Mount 1/4-28 UNF to Male Luer
|Heparin sodium salt from porcine intestinal mucosa
|Luer-Stub Adapter BD Intramedic 20 Gauge
|Male Luer (2) to Female Luer Thread Style Tee
|Nalgene 180 Clear Plastic PVC Tubing
|Paraformaldehyde Aqueous Solution - 32%
|Electron Microscopy Sciences
|Fixative solution. Diluted to 4% with phosphate buffered saline
|Permatex Ultra Blue Multipurpose RTV Silicone Gasket Maker
|Silicone Gasket Maker for air-tight sealing of chambers
|Phosphate-Buffered Saline, 1x Without Calcium and Magnesium
|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
|Animal processing container
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