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 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.
A variety of mouse models of lung oncogenesis and cancer metastasis to the lung exist ranging from complex GEMMs to carcinogen-induced models to syngeneic and xenograft models, where cancer cells are injected via intracardiac, intrathoracic, the tail vein, or other methods to establish tumors within the lung. All these models share the common need for histological evaluation of lung histology and pathology. Thus, it is necessary to have a robust yet rapid method to perform necropsies of mice while perfusing the lungs to remove excess blood, and inflating and fixing the lungs to clearly visualize lung architecture. Speed is a critical component of this procedure as it may be necessary to collect the lungs from dozens of mice at a single time point. This procedure can be performed in less than 6 minutes per mouse.
While this procedure is more than sufficient for evaluating tumor histology, it is not recommended for those who wish to perform stereology or morphometric measurements of the lungs. Such measurements require lung inflation to be standardized, as does the calculation of absolute surface area of the lung, absolute volume, and alveolar size and number1. This method is also not optimal for some imaging approaches. For example, imaging of the lungs via µCT for ex vivo morphometric analysis requires that the lungs remain filled with air2. When the preservation of air spaces and dimensions are the primary concern, it is recommended to fix the lungs by perfusion dehydration techniques3,4. One of the biggest concerns of this model is the potential for rupturing of the alveolar walls, lessening its use in studies of emphysema; however, the recommended procedure for fixation of lungs for the study of emphysema is still quite similar, as it is recommended to fix the lungs either by intratracheal instillation of 10% formalin (similar to the protocol described here) under constant fluid pressure or by in situ fixation5.
The advantage of the described procedure here is that it does not require constant fluid pressure, instead inflating the lungs until they have fully expanded, thus decreasing the time needed for the procedure. The procedure here described closely resembles the methods recommended by an armamentarium of the Society of Toxicologic Pathology, where a subcommittee was formed to recommend the best methods of lung fixation for toxicology studies. The majority of scientists within this subcommittee recommended fixing the lungs by intratracheal instillation with a syringe, though there were varying recommendations on the time the lung was left in the fixative6. Thus, while a variety of methods of lung inflation and fixation exist, the method described herein is proposed to be the optimal method to quickly inflate and fix the lungs for downstream tumor histological evaluation.
All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham.
1. Experimental protocol
The above protocol allows for quick perfusion, inflation, and fixation of mouse lungs. The figures shown below represent the importance of each step. Figure 1 depicts H&E stained lungs that have been perfused with PBS and lungs in which the perfusion step has been skipped or the lungs failed to perfuse correctly. As shown, excess blood in the poorly perfused lungs creates less than ideal histology and can make it challenging to fully observe lung architecture. Figure 2 demonstrates both the importance of inflation as well as the dangers of over-inflation. It is more difficult to identify areas of hyperplasia in un-inflated lungs due to the compression and close proximity of the alveoli; however, in the overinflated lungs, many of the alveolar walls have been broken and this could be mistaken for emphysema if not careful. Figure 3 depicts tumor histology in lungs which have been perfused and inflated using the technique described herein.
Figure 1: Representative H&E staining of perfused and non-perfused lungs. (A) Lungs perfused with PBS. (B) Lungs not perfused. Please click here to view a larger version of this figure.
Figure 2: Representative H&E staining of inflated, uninflated, and overinflated lungs. (A) Lungs were inflated and fixed with 10% formalin until the lungs fully expanded. (B) Lungs were not inflated through the trachea and instead directly placed in 10% formalin. (C) Lungs were inflated and fixed with 10% formalin but 10% formalin was continuously pushed into the lungs past full expansion resulting in over-inflation. Please click here to view a larger version of this figure.
Figure 3: Representative H&E staining of perfused and inflated mouse lungs. (A) Lung tumors were induced using the chemical carcinogen urethane. (B) Spontaneous lung metastases from a human xenografted cell line Please click here to view a larger version of this figure.
The procedure described above for the perfusion, inflation, and fixation of mouse lungs is ideal for quick and efficient preparation of mouse lungs for lung tumor histology and pathology analysis. The procedure does not require any special equipment and can be performed in less than 6 minutes per mouse. The procedure does not require a fixed volume for inflation nor constant fluid pressure. Because this procedure is not standardized, it is not recommended for those wishing to perform stereological or morphometric analyses of the lung. Procedures where such standardization is required have been better described1,7.
The most critical steps of this protocol are perfusion and inflation. It is important to perfuse through the right ventricle of the heart, whereas if perfusion is done through the left ventricle, the lungs will not perfuse. It is easy to tell if perfusion is done correctly as the lungs will turn white. Instillation of fixative through the trachea allows inflation of the lungs, which allows for easier downstream histological analysis of lung architecture. It is important to stop administration of fixative as soon as the lungs have fully expanded, as over-inflation can cause alveolar wall breakage and the appearance of emphysema. Once the lungs are inflated, some formalin will backflow out of the trachea. This is normal and does not impact downstream histological analysis; however, if this is a concern the trachea can be tied off prior excising the lungs.
While this protocol uses 10% buffered formalin to fix the lungs, which is the most commonly recommended fixative5,6, there are reports of artifacts introduced by this fixative, namely shrinkage of the lung tissue1,8. If this is a concern, follow the guidelines of the American Thoracic Society and European Respiratory Society for the assessment of lung structure1. Another potential fixative not commonly recommended but that may prove useful is Bouin’s solution, which may provide better contrast for the evaluation of lung surface nodules9,10. In summary, the herein described protocol provides a robust and simple method for the fixation of mouse lungs for tumor histology.
The authors have nothing to disclose.
Research reported in this publication was supported by the National Center for Advancing Translational Sciences under award number UL1TR003096 (MDE), National Heart, Lung, and Blood Institute Predoctoral Fellowship in Lung Diseases Training Program 5T32HL134640 (MLD).
10% buffered formalin | Fisher | 23-245685 | |
22 G Needle | BD | 305155 | |
3 mL syringe | BD | 309656 | |
70% Ethanol | Decon | 2405 | |
Forceps | Harvard Apparatus | 72-8595 | |
Heparin | Fisher | H19 | |
Phosphate Buffered Saline (PBS) | Corning | 21-030-CV | |
Surgical scissors | Harvard Apparatus | 72-8428 |