Name: Perfusion and Inflation of the Mouse Lung for Tumor Histology (Video) | JoVE Journal
Uploaded: 2020-08-06T15:30:00.000+00:00
Duration: 4 min 24 s
Description: Learn perfusion and inflation of mouse lungs for tumor histology using this reliable fixation method. This technique enables accurate pathological examination and metastasis evaluation in cancer research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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. Next, insert surgical scissors into the small hole created from incision and cut vertically up the center midline to just below the neck of the mouse.</li>
	<li>Pull the skin back with fingers and inspect the axillary lymph nodes.</li>
	<li>Using surgical scissors, make a 3.5 mm lateral incision to open the abdominal cavity, and then cut in the anterior direction up to the bottom of the thorax. Inspect the organs in the abdominal cavity: liver, spleen, kidneys, etc.</li>
	<li>With the flat of the surgical scissors, or using forceps, move the liver to expose the diaphragm. Inspect the diaphragm for tumor growth or metastases. Then, gently snip the diaphragm on the operator&#8217;s right side, allowing it to expand. Gently cut the diaphragm from right to left to expose the thoracic cavity and lungs. Be careful not to cut the lungs.</li>
	<li>Cut up through the lateral extreme of the left rib cage (on operator&#8217;s right) to inspect the left lobe of the lungs.</li>
	<li>Gently move the right lobes of the lung out of the way and cut up the lateral extreme of the right rib cage and remove rib cage. 
	NOTE: Removal of the rib cage is optional, though removal enables clearer view of later lung inflation.
	<ol>
		<li>If fresh or frozen lung tissue is required, use hemostat forceps to clamp the bronchus of the left lobe and resect the left lung using surgical scissors prior to perfusion.</li>
	</ol>
	</li>
	<li>Using the forceps to lift the tissue covering the trachea, cut away any excess tissue. Then gently cut the thin tissue lining the trachea to expose the airway.</li>
	<li>Cut through the renal artery with surgical scissors.</li>
	<li>To perfuse the lungs, use a 3 mL syringe with a 22 G needle to inject 1x PBS with 10 U/mL heparin into the right ventricle of the heart. Slowly perfuse the lungs at approximately 300 &#181;L/s with PBS/heparin. The lungs will frequently turn white. 2.5 mL of PBS are generally used in this step.</li>
	<li>For lung inflation, use a 3 mL syringe with a 22 G needle, this time held parallel to the trachea. Insert the needle into the trachea and inject 10% formalin with rate of flow no greater than ~200 &#181;L/s 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.
	<ol>
		<li>(Optional) Prior to withdrawing needle and lung inflation, use suture thread to tie off the trachea. To achieve this, use 4 inches of suture thread holding the point of the thread with a small pair of forceps. Place the thread on the dorsal side of the trachea and pull through to make a loop around the needle. Next make an overhand knot around the needle. Pull knot tight, remove needle from trachea, close knot.</li>
	</ol>
	</li>
	<li>Use forceps to lift the heart, insert surgical scissors directly behind the lungs, and cut connective tissue while the lifting the heart to resect lungs.</li>
	<li>Cut the heart to remove it from lungs.</li>
	<li>Place the lungs a cassette labeled with the mouse ID or study ID. Place the cassette in 10% buffered formalin and fix for 24-48 h. Lungs can be left in fixative for over a year if desired.</li>
	<li>Transfer the cassette containing the lungs to 70% ethanol and proceed to processing for histology.</li>
</ol>

<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/pubmed?term=((An+official+research+policy+statement+of+the+American+Thoracic+Society%2FEuropean+Respiratory+Society%3A+standards+for+quantitative+assessment+of+lung+structure%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+and+Critical+Care+Medicine%22%5BJournal%5D)">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/pubmed?term=((Optimized+murine+lung+preparation+for+detailed+structural+evaluation+via+micro-computed+tomography%5BTitle%5D)+AND+%22Journal+of+Applied+Physiology%22%5BJournal%5D)">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/pubmed?term=((Lung+fixation+for+the+preservation+of+air+spaces%5BTitle%5D)+AND+%22Experimental+Lung+Research%22%5BJournal%5D)">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/pubmed?term=((Perfusion+dehydration+fixes+elastin+and+preserves+lung+air-space+dimensions%5BTitle%5D)+AND+%22Journal+of+Applied+Physiology%22%5BJournal%5D)">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/pubmed?term=((A+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">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/pubmed?term=((Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies%5BTitle%5D)+AND+%22Toxicologic+Pathology%22%5BJournal%5D)">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/pubmed?term=((Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Effects+of+10%25+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.+A+comparison+of+eleven+laboratory+species%5BTitle%5D)+AND+%22American+Review+of+Respiratory+Disease%22%5BJournal%5D)">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/pubmed?term=((MicroRNA-31+initiates+lung+tumorigenesis+and+promotes+mutant+KRAS-driven+lung+cancer%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">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/pubmed?term=((Wogonin+suppresses+melanoma+cell+B16-F10+invasion+and+migration+by+inhibiting+Ras-medicated+pathways%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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

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&amp;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...

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 ...

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

Research

JoVE Journal

Cancer Research

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

This video describes a method of preparing lungs for ex vivo live imaging to study interactions between cancer cells and stromal cells, using agarose for inflation. In the sample protocol, agarose inflation procedure is described before observing live cellular interactions.

Whole Lung Agarose Inflation: A Method of Preparing  Lungs for Ex vivo Live Imaging

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

The goal of this paper is to describe simple methods that will greatly aid in the setup and analysis of mouse lungs with lung cancer or other pathologies. We present 3 protocols to simply and reliably carry out lung instillations, fixation, and lung volume measurements.

Instillation and Fixation Methods Useful in Mouse Lung Cancer Research

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

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.

Air-Inflation of Murine Lungs with Vascular Perfusion-Fixation

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

We present a method for a fungal pathogenesis model that preserves the natural positioning of fungal spores in the lung airways for analysis via fluorescent microscopy.

Efficient Method for Imaging Murine Lungs that Preserves Spatial Dynamics of Fungal Spores in the Airways

A major challenge of developing new therapies for lung cancer includes modeling the tumor microenvironment as regional disease progresses to metastatic disease. Here, we describe a simple method of establishing an orthotopic syngeneic model of lung cancer that allows for in vivo tracking of disease over time.

A Minimally Invasive Method for Generating a Syngeneic Orthotopic Mouse Model of Lung Cancer

Learn perfusion and inflation of mouse lungs for tumor histology using this reliable fixation method. This technique enables accurate pathological examination and metastasis evaluation in cancer research.

Perfusion and Inflation of the Mouse Lung for Tumor Histology

Explore More Videos