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).

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
<ol>
	<li>Sacrifice the mouse using an approved IACUC method. Here, we used cervical dislocation of a mouse anesthetized with 5% isoflurane. Use an appropriate mouse for the study; here, we use an 8-week old FVB mouse</li>
	<li>Using surgical scissors, make a 3.5-5 mm horizontal incision in the middle of lower abdomen. N...

<ol>
	<li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R., Structure, A. <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>Vasilescu, D. M., Knudsen, L., Ochs, M., Weibel, E. R., Hoffman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+murine+lung+preparation+for+detailed+structural+evaluation+via+micro-computed+tomography.">Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography.</a> Journal of Applied Physiology. 112 (1), 159-166 (2012).</li><li>Blumler, P., Acosta, R. H., Thomas-Semm, A., Reuss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fixation+for+the+preservation+of+air+spaces.">Lung fixation for the preservation of air spaces.</a> Experimental Lung Research. 30 (1), 73-82 (2004).</li><li>Oldmixon, E. H., Suzuki, S., Butler, J. P., Hoppin, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+dehydration+fixes+elastin+and+preserves+lung+air-space+dimensions.">Perfusion dehydration fixes elastin and preserves lung air-space dimensions.</a> Journal of Applied Physiology. 58 (1), 105-113 (1985).</li><li>Braber, S., Verheijden, K. A., Henricks, P. A., 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>Renne, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies.">Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies.</a> Toxicologic Pathology. 29 (5), 587-589 (2001).</li><li>Limjunyawong, N., Mock, J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research.">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> Journal of Visualized Experiments. (102), e52964 (2015).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+10%+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.+A+comparison+of+eleven+laboratory+species.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage. A comparison of eleven laboratory species.</a> American Review of Respiratory Disease. 132 (5), 1078-1083 (1985).</li><li>Edmonds, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-31+initiates+lung+tumorigenesis+and+promotes+mutant+KRAS-driven+lung+cancer.">MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer.</a> Journal of Clinical Investigation. 126 (1), 349-364 (2016).</li><li>Zhao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wogonin+suppresses+melanoma+cell+B16-F10+invasion+and+migration+by+inhibiting+Ras-medicated+pathways.">Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.</a> PLoS One. 9 (9), 106458 (2014).</li></ol>

The authors have nothing to disclose.

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 analyse...

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 &#181;CT 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.

<table>
	<tbody>
		<tr>
			<td>10% buffered formalin</td>
			<td>Fisher</td>
			<td>23-245685</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Needle</td>
			<td>BD</td>
			<td>305155</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon</td>
			<td>2405</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Harvard Apparatus</td>
			<td>72-8595</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisher</td>
			<td>H19</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
	</tbody>
</table>

perfusion and inflation of the mouse lung for tumor histology

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.

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&#38;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...

Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

Perfusion and Inflation of the Mouse Lung for Tumor Histology

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 ...

Demonstrating the procedure will be Mackenzie Davenport, a graduate student from the Edmonds Laboratory. After euthanizing the mouse, use surgical scissors to make a 3.5 to five millimeter horizontal incision in the middle of the lower abdomen. Next, insert surgical scissors into the incision and cut vertically up the center midline to just below the neck of the mouse.Pull the skin back with fingers and inspect the axillary lymph nodes. Use surgical scissors to make a 3.5 millimeter lateral incision to open the abdominal cavity. Then cut in the interior direction up to the bottom of the thorax.Inspect the organs in the abdominal cavity such as the liver, spleen, and kidneys. With the flat of the surgical scissors move the liver to expose the diaphragm. Inspect the diaphragm for tumor growth or metastases, then gently snip the diaphragm on the operator's right side, allowing it to expand.Cut the diaphragm from right to left to expose the thoracic cavity and lungs, taking care not to cut the lungs. Cut up through the lateral extreme of the left rib cage to inspect the left lobe of the lungs. Gently move the right lobes of the lung out of the way.Then cut up the lateral extreme of the right rib cage and remove the rib cage. If fresh or frozen lung tissue is required, use hemostat forceps to clamp the bronchus of the left lobe and resect the left lung with surgical scissors prior to perfusion. Use the forceps to lift the tissue covering the trachea and cut away any excess tissue, then gently cut the thin tissue lining the trachea to expose the airway.Cut through the renal artery with surgical scissors. To perfuse the lungs, use a three milliliter syringe with a 22 gauge needle to inject PBS with 10 units per milliliter heparin into the right ventricle of the heart. Slowly profuse the lungs at approximately 300 microliters per second causing the lungs to turn white.For lung inflation, use a three milliliter syringe with a 22 gauge needle and hold it parallel to the trachea. Insert the needle into the trachea and inject 10%formalin with a flow rate no greater than 200 microliters per second until the lungs have fully inflated. Once the lungs are inflated, formalin will backflow out of the trachea.Hold the needle in place for a few more seconds and then withdraw. Use forceps to lift the heart, insert surgical scissors directly behind the lungs and cut the connective tissue to resect to the lungs. Then cut the to remove it from the lungs.Place the lungs in a cassette labeled with the mouse ID or study ID, then place the cassette in 10%buffered formalin for 24 to 48 hours. Lungs can be left in fixative for over a year if desired. When finished, transfer the cassette containing the lungs to 70%ethanol and proceed with histology.This protocol allows for quick perfusion, inflation, and fixation of mouse lungs. Tumor histology in lungs which have been perfused and inflated using this technique is demonstrated here. When the profusion step is skipped, or the lungs fail to perfuse correctly, excess blood in the lungs creates less than ideal histology and can make it challenging to fully observe lung architecture.Inflation is an important step in this protocol. It is more difficult to identify areas of hyperplasia in uninflated lungs due to the compression and close proximity of the alveoli. However, in overinflated lungs, many of the alveolar walls have been broken which could be mistaken for emphysema.

perfusion-and-inflation-of-the-mouse-lung-for-tumor-histology

Research

JoVE Journal

Cancer Research

16.9K Görüntüleme.  University of Alabama at Birmingham. Demonstrating the procedure will be Mackenzie Davenport, a graduate student from the Edmonds Laboratory. After euthanizing the mouse, use surgical scissors to make a 3.5 to five millimeter horizontal incision in the middle of the lower abdomen. Next, insert surgical scissors into the incision and cut vertically up the center midline to just below the neck of the mouse.Pull the skin back with fingers and inspect the axillary lymph nodes. Use surgical scissors to make a 3.5 millimeter la...