Name: perfusion and inflation of the mouse lung for tumor histology
Uploaded: 2020-08-06T15:30:00
Description: Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

Forskning rapporteret i denne publikation blev støttet af National Center for Advancing Translational Sciences under tildeling nummer UL1TR003096 (MDE), National Heart, Lung, og Blood Institute Predoctoral Fellowship i lungesygdomme Training Program 5T32HL134640 (MLD).

Alle de metoder, der er beskrevet her, er blevet godkendt af Institutional Animal Care and Use Committee (IACUC) ved University of Alabama i Birmingham.
1. Eksperimentel protokol
<ol><li>Ofre musen ved hjælp af en godkendt IACUC metode. Her brugte vi cervikal dislokation af en mus bedøvet med 5% isoflurane. Brug en passende mus til undersøgelsen. her bruger vi en 8 uger gammel FVB-mus</li>
	<li>Brug kirurgisk saks, lav et 3,5-5 mm vandret snit midt i underlivet. Derefter indsætte kirurgi...

<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>

Den procedure, der er beskrevet ovenfor for perfusion, inflation og fiksering af muse lunger, er ideel til hurtig og effektiv forberedelse af muse lunger til lungetumor histologi og patologianalyse. Proceduren kræver ikke noget specielt udstyr og kan udføres på mindre end 6 minutter pr. Mus. Proceduren kræver ikke et fast volumen for inflation eller konstant væsketryk. Da denne procedure ikke er standardiseret, anbefales det ikke til dem, der ønsker at udføre stereologiske eller morfometriske analyser af lungerne....

En række musemodeller af lunge oncogenesis og kræft metastaser til lungerne findes lige fra komplekse GEMMs til kræftfremkaldende-induceret modeller til syngeneic og xenograft modeller, hvor kræftceller injiceres via intracardiac, intrathoracic, halen vene, eller andre metoder til at etablere tumorer i lungerne. Alle disse modeller deler det fælles behov for histologisk evaluering af lunge histologi og patologi. Således er det nødvendigt at have en robust, men hurtig metode til at udføre obduktioner af mus, mens perfusing lungerne for at fjerne overskydende blod, og oppustning og fastsættelse af lungerne til klart at visualisere lunge arkitektur. Hastighed er en kritisk komponent i denne procedure, da det kan være nødvendigt at indsamle lungerne fra snesevis af mus på et enkelt tidspunkt. Denne procedure kan udføres på mindre end 6 minutter pr. Mus.
Mens denne procedure er mere end tilstrækkelig til at evaluere tumor histologi, anbefales det ikke til dem, der ønsker at udføre stereologi eller morfometriske målinger af lungerne. Sådanne målinger kræver, at lungeinflationen standardiseres, og det samme gælder beregningen af lungernes absolutte overfladeareal, absolutte volumen og alveolarstørrelse og nummer1. Denne metode er heller ikke optimal for nogle billedbehandlingsmetoder. For eksempel kræver billeddannelse af lungerne via μCT til ex vivo morfometrisk analyse, at lungerne forbliver fyldt med luft2. Når bevarelsen af luftrum og dimensioner er den primære bekymring, anbefales det at rette lungerne ved perfusion dehydreringsteknikker3,4. En af de største bekymringer ved denne model er potentialet for brud på alveolarvæggene, der mindsker brugen i undersøgelser af emfysem; den anbefalede procedure for fiksering af lunger til undersøgelse af emfysem er dog stadig ret ens, da det anbefales at fastgøre lungerne enten ved intratracheal instillation af 10% formalin (svarende til den protokol, der er beskrevet her) under konstant væsketryk eller ved in situ-fiksering5.
Fordelen ved den beskrevne procedure her er, at det ikke kræver konstant væsketryk, i stedet oppuste lungerne, indtil de er fuldt udvidet, hvilket reducerer den tid, der er nødvendig for proceduren. Proceduren her beskrevet ligner de metoder, der anbefales af et oprustningsarium fra Society of Toxicologic Pathology, hvor et underudvalg blev dannet for at anbefale de bedste metoder til lungefiksering til toksikologiske undersøgelser. De fleste forskere i dette underudvalg anbefalede at fastgøre lungerne ved intratracheal instillation med en sprøjte, selvom der var forskellige anbefalinger om det tidspunkt, hvor lungen blev efterladt ifikseringstiden 6. Således, mens en række metoder til lunge inflation og fiksering findes, den metode, der er beskrevet heri foreslås at være den optimale metode til hurtigt at puste og fastsætte lungerne for downstream tumor histologiske evaluering.

<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

Evnen til at evaluere lunge histologi er afgørende for områderne lungekræft forskning og kræft metastaser. Det er lige så vigtigt at udføre obduktioner hurtigt og effektivt fra undersøgelser uden at ofre kvaliteten af de indkøbte væv. Målet med denne protokol er at præsentere en metode til hurtigt at gennemgå, puste og fastsætte mus lunger til downstream histologiske analyse. Denne metode standardiserer ikke lungeinflationen; således kræver det ikke nogen særlige procedurer eller udstyr og i stedet blot instills fikseringsmiddel direkte gennem luftrøret efter perfusion gennem hjertet. Dette giver mulighed for tilstrækkelig vurdering af tumor størrelse, histologi, og scoring. Dette giver også mulighed for indsamling af frosset væv forud for lungevæv fiksering. Denne metode er begrænset, idet den ikke giver mulighed for senere morfometrisk kvantificering af lungerne; Det er dog mere end tilstrækkeligt til lungetumoranalyse fra genetisk manipulerede musemodeller (GEMMs), syngeneiske modeller samt xenograft tumor- og metastaseundersøgelser.

Ovenstående protokol giver mulighed for hurtig perfusion, inflation og fiksering af muse lunger. Nedenstående tal viser betydningen af hvert trin. Figur 1 viser H&E farvede lunger, der er blevet perfunderet med PBS og lunger, hvor perfusionstrinnet er sprunget over, eller lungerne ikke har perfuse korrekt. Som vist skaber overskydende blod i de dårligt perfunderede lunger mindre end ideel histologi og kan gøre det udfordrende at fuldt ud observere lungearkitektur. Fi...

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

Formålet med denne metode er at præsentere en enkel og effektiv metode til perfusion, inflation og fiksering af muse lunger til undersøgelse af lungetumorpatologi og evaluering af metastaser til lungerne.

Perfusion og inflation af musens lunge for tumor histologi

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.

Research

JoVE Journal

Cancer Research

Lung Tumor Cell Recruitment Assay

Lunge Tumor celle rekruttering Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Det<em&gt; Drosophila</em&gt; Imaginære Disc Tumor Model: Visualisering og Kvantificering af genekspression og tumorindtrængen Brug Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Den Colon-26 Carcinoma Tumor-bærende mus som en model for Studiet af Cancer Kakeksi

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Target Cell pre berigelse og hele genom forstærkning for enkelt celle Downstream karakterisering

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Etablering af en lunge kolonisering Assay for Cirkulerende Tumor Cell visualisering i lungevæv

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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

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

Udnytte 18F-FDG PET/CT billeddannelse og kvantitative histologi at måle dynamiske ændringer i glukose stofskifte i musemodeller af lungekræft

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

Acellulær og cellulære Lung Model til at studere Tumor metastaser

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

Digital Polymerase Chain Reaction Assay for den genetiske Variation i en sporadisk familiær adenomatøs polypose Patient med formatet Chip i rør

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

Påvisning af lungetumorprogression hos mus ved ultralydsscanning

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Patologisk analyse af lungemetasase Efter Lateral Hale-Vene Injektion af tumorceller

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...