The authors would like to thank Drs. Yuan Hao and Kaifeng Liu for their technical support. This work was supported by an NIH 1R01 HL150106-01A1, the Parker B. Francis Fellowship, and the Pulmonary Hypertension Association Aldrighetti Research Award to Dr. Ke Yuan.

All animal care was in accordance with the guidelines of Boston Children&#39;s Hospital and the Institutional Animal Care and Use Committee approved protocols. The mice used in this study are wild type C57/B6 mice and Cdh5-CreERT2 x Ai14 tdTomato crossed mice.
1. Preparation of solutions
<ol>
	<li>Prepare phosphate buffer solution (1x PBS) and 2% agarose solution required during the experiment in advance.
	<ol>
		<li>Mix 2 g agarose powder into 100 mL of autoclaved water. Heat it in the micr...

<ol>
	<li>Sparrow, D., Weiss, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+physiology.">Respiratory physiology.</a> Annual Review of Gerontology &amp; Geriatrics. 6, 197-214 (1986).</li><li>Gerckens, M., 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=Generation+of+human+3D+lung+tissue+cultures+(3D-LTCs)+for+disease+modeling.">Generation of human 3D lung tissue cultures (3D-LTCs) for disease modeling.</a> Journal of Visualized Experiments: JoVE. (144), e58437 (2019).</li><li>Li, G., 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=Preserving+airway+smooth+muscle+contraction+in+precision-cut+lung+slices.">Preserving airway smooth muscle contraction in precision-cut lung slices.</a> Scientific Reports. 10 (1), 6480 (2020).</li><li>Rosales Gerpe, M. C., 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=Use+of+precision-cut+lung+slices+as+an+ex+vivo+tool+for+evaluating+viruses+and+viral+vectors+for+gene+and+oncolytic+therapy.">Use of precision-cut lung slices as an ex vivo tool for evaluating viruses and viral vectors for gene and oncolytic therapy.</a> Molecular Therapy: Methods &amp; Clinical Development. 10, 245-256 (2018).</li><li>Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+lung+physiology+in+health+and+disease+with+lung+slices.">Exploring lung physiology in health and disease with lung slices.</a> Pulmonary Pharmacology &amp; Therapeutics. 24 (5), 452-465 (2011).</li><li>Liu, 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=Mouse+lung+slices:+An+ex+vivo+model+for+the+evaluation+of+antiviral+and+anti-inflammatory+agents+against+influenza+viruses.">Mouse lung slices: An ex vivo model for the evaluation of antiviral and anti-inflammatory agents against influenza viruses.</a> Antiviral Research. 120, 101-111 (2015).</li><li>de Graaf, I. A., 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=Preparation+and+incubation+of+precision-cut+liver+and+intestinal+slices+for+application+in+drug+metabolism+and+toxicity+studies.">Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies.</a> Nature Protocols. 5 (9), 1540-1551 (2010).</li><li>Alsafadi, H. N., 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=Applications+and+approaches+for+three-dimensional+precision-cut+lung+slices.+Disease+modeling+and+drug+discovery.">Applications and approaches for three-dimensional precision-cut lung slices. Disease modeling and drug discovery.</a> American Journal of Respiratory Cell and Molecular Biology. 62 (6), 681-691 (2020).</li><li>Morin, J. P., 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=Precision+cut+lung+slices+as+an+efficient+tool+for+in+vitro+lung+physio-pharmacotoxicology+studies.">Precision cut lung slices as an efficient tool for in vitro lung physio-pharmacotoxicology studies.</a> Xenobiotica. 43 (1), 63-72 (2013).</li><li>Springer, J., Fischer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substance+P-induced+pulmonary+vascular+remodelling+in+precision+cut+lung+slices.">Substance P-induced pulmonary vascular remodelling in precision cut lung slices.</a> The European Respiratory Journal. 22 (4), 596-601 (2003).</li><li>Suleiman, S., 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=Argon+reduces+the+pulmonary+vascular+tone+in+rats+and+humans+by+GABA-receptor+activation.">Argon reduces the pulmonary vascular tone in rats and humans by GABA-receptor activation.</a> Scientific Reports. 9 (1), 1902 (2019).</li><li>Rieg, A. 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=Cardiovascular+agents+affect+the+tone+of+pulmonary+arteries+and+veins+in+precision-cut+lung+slices.">Cardiovascular agents affect the tone of pulmonary arteries and veins in precision-cut lung slices.</a> PLoS One. 6 (12), 29698 (2011).</li><li>Perez, J. F., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+frequency+of+calcium+oscillations+induced+by+5-HT,+ACH,+and+KCl+determine+the+contraction+of+smooth+muscle+cells+of+intrapulmonary+bronchioles.">The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles.</a> The Journal of General Physiology. 125 (6), 535-553 (2005).</li><li>Deng, C. Y., 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=Upregulation+of+5-hydroxytryptamine+receptor+signaling+in+coronary+arteries+after+organ+culture.">Upregulation of 5-hydroxytryptamine receptor signaling in coronary arteries after organ culture.</a> PLoS One. 9 (9), 107128 (2014).</li><li>Sandker, S. C., 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=Adventitial+dissection:+A+simple+and+effective+way+to+reduce+radial+artery+spasm+in+coronary+bypass+surgery.">Adventitial dissection: A simple and effective way to reduce radial artery spasm in coronary bypass surgery.</a> Interactive Cardiovascular and Thoracic Surgery. 17 (5), 784-789 (2013).</li><li>Naik, J. S., 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=Pressure-induced+smooth+muscle+cell+depolarization+in+pulmonary+arteries+from+control+and+chronically+hypoxic+rats+does+not+cause+myogenic+vasoconstriction.">Pressure-induced smooth muscle cell depolarization in pulmonary arteries from control and chronically hypoxic rats does not cause myogenic vasoconstriction.</a> Journal of Applied Physiology. 98 (3), 1119-1124 (2005).</li><li>Lopez-Lopez, J. G., 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=Diabetes+induces+pulmonary+artery+endothelial+dysfunction+by+NADPH+oxidase+induction.">Diabetes induces pulmonary artery endothelial dysfunction by NADPH oxidase induction.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 295 (5), 727-732 (2008).</li><li>Gonzalez-Tajuelo, 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=Spontaneous+pulmonary+hypertension+associated+with+systemic+sclerosis+in+P-selectin+glycoprotein+Ligand+1-deficient+mice.">Spontaneous pulmonary hypertension associated with systemic sclerosis in P-selectin glycoprotein Ligand 1-deficient mice.</a> Arthritis &amp; Rheumatology. 72 (3), 477-487 (2020).</li><li>Bai, Y., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+Ca2++sensitivity+of+airway+smooth+muscle+cells+in+murine+lung+slices.">Modulation of the Ca2+ sensitivity of airway smooth muscle cells in murine lung slices.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 291 (2), 208-221 (2006).</li><li>Nishiyama, S. 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=Vascular+function+and+endothelin-1:+tipping+the+balance+between+vasodilation+and+vasoconstriction.">Vascular function and endothelin-1: tipping the balance between vasodilation and vasoconstriction.</a> Journal of Applied Physiology. 122 (2), 354-360 (2017).</li><li>Schneider, M. P., Inscho, E. W., Pollock, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+vasoconstrictor+responses+to+endothelin+in+afferent+arterioles+during+a+high-salt+diet.">Attenuated vasoconstrictor responses to endothelin in afferent arterioles during a high-salt diet.</a> American Journal of Physiology. Renal Physiology. 292 (4), 1208-1214 (2007).</li><li>Inscho, E. W., Imig, J. D., Cook, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Afferent+and+efferent+arteriolar+vasoconstriction+to+angiotensin+II+and+norepinephrine+involves+release+of+Ca2++from+intracellular+stores.">Afferent and efferent arteriolar vasoconstriction to angiotensin II and norepinephrine involves release of Ca2+ from intracellular stores.</a> Hypertension. 29, 222-227 (1997).</li><li>Vecchione, C., 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=Protection+from+angiotensin+II-mediated+vasculotoxic+and+hypertensive+response+in+mice+lacking+PI3Kgamma.">Protection from angiotensin II-mediated vasculotoxic and hypertensive response in mice lacking PI3Kgamma.</a> The Journal of Experimental Medicine. 201 (8), 1217-1228 (2005).</li></ol>

In this manuscript, an enhanced method to produce high-resolution images of murine lung tissue that preserves the vascular structure and optimizes experimental flexibility is described, specifically using the application of PCLS to obtain microslices of lung tissue that can be viewed in three dimensions with preserved contractility of the vasculature. Using the viability reagent, the protocol demonstrates that carefully prepared and preserved slices can retain viability for more than a week. Preserved viability of the mi...

Advances in the preparation and imaging of lung tissue that preserves cellular components without sacrificing anatomical structure provide a detailed understanding of pulmonary diseases. The ability to identify proteins, RNA, and other biological compounds while maintaining physiological structure offers vital information on the spatial arrangement of cells that can broaden the understanding of the pathophysiology in numerous pulmonary diseases. These detailed images can lead to a better understanding of pulmonary vascular diseases, such as pulmonary artery hypertension, when applied to animal models, potentially leading to improved therapeutic strategies.
Despite advances in technology, obtaining high-quality images of murine lung tissue remains a challenge. The respiratory cycle is driven by a negative intrathoracic pressure generated during inhalation1. When traditionally obtaining biopsies and preparing lung samples for imaging, the negative pressure gradient is lost resulting in the collapse of the airway and vasculature, which no longer represents itself in its present state. To achieve realistic images reflective of current conditions, the pulmonary airways must be reinflated, and the vasculature perfused, changing the dynamic lung into a static fixture. The application of these distinct techniques allows preservation of structural integrity, pulmonary vasculature, and cellular components, including immune cells such as macrophages, allowing lung tissue to be viewed as close to its physiological state as possible.
Precision cut lung slicing (PCLS) is an ideal tool for studying the anatomy and physiology of pulmonary vasculature2. PCLS provides detailed imaging of the lung tissue in three dimensions while preserving structural and cellular components. PCLS has been used in animal and human models to allow for live, high-resolution images of cellular functions in three dimensions, making it an ideal tool to study potential therapeutic targets, measure small airway contraction and study the pathophysiology of chronic lung diseases such as COPD, ILD, and lung cancer3. Using similar techniques, the exposure of PCLS samples to vasoconstrictors can preserve lung structure and vessel contractility, replicating in vitro conditions. Along with preserving contractility, prepared samples can undergo additional analysis such as RNA sequencing, Western blot, and flow cytometry when prepared correctly. Finally, reporter color labeled cells marked with tdTomato fluorescence after lung harvest can preserve labeling after preparing microslices, making it ideal for cell tracking studies. The integration of these techniques provides a sophisticated model preserving the spatial arrangement of cells and vessel contractility that can lead to a more detailed understanding of the signaling cascades and potential therapeutic options in pulmonary vasculature disease.
In this manuscript, PCLS murine lung tissue is exposed to vasoconstrictors, demonstrating preserved structural integrity and vessel contractility. The study demonstrates that the tissue prepared and handled appropriately can remain viable for 10 days. The study also demonstrates the preservation of cells with endogenous fluorescence (tdTomato), allowing samples to provide high-resolution images of the pulmonary vasculature and architecture. Finally, ways to handle and prepare tissue slices for RNA measurement and Western blot to investigate underlying mechanisms have been described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5cc of fractionated heparin in syringe</td>
			<td>BD</td>
			<td>100 USP units per mL</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Corning</td>
			<td>&#160;21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>20 1/2 inch gauge blunt end needle for trachea cannulation</td>
			<td>Cml Supply</td>
			<td>90120050D</td>
			<td></td>
		</tr>
		<tr>
			<td>30cc syringe</td>
			<td>BD</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti Anti solution</td>
			<td>Gibco</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated vibrating blade microtome</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Viability Reagent (alamarBlue)</td>
			<td>Thermofisher</td>
			<td>DAL1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal</td>
			<td>Zeiss</td>
			<td>880</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium and GLutaMAX, supplemented with 10% FBS, 1% Pen/Strep</td>
			<td>Gibco</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelin-1</td>
			<td>Sigma</td>
			<td>E7764</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar and Pestle</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis and extraction buffer</td>
			<td>Thermoscientific</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical suture 6/0</td>
			<td>FST</td>
			<td>18020-60</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen, Thermofisher</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Low Melting Point Agarose</td>
			<td>Invitrogen</td>
			<td>16520050</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica Biosystems</td>
			<td>VT1200 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Winged blood collection set (Butterfly needle) 25-30G</td>
			<td>BD</td>
			<td>25-30G</td>
			<td></td>
		</tr>
	</tbody>
</table>

precision cut lung slices as an efficient tool for ex vivo pulmonary vessel structure and contractility studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

When added to cells or tissue, the viability reagent is modified by the reducing environment of viable tissue and turns pink/red, becoming highly fluorescent. The representative color changes detected from day 0-1 and day 9-10 are demonstrated in Figure 3. As noted, the solution started blue and turned pink overnight, demonstrating viability. Color change typically occurs within 1-4 h; however, a longer time may be necessary. To assay for viability, a plate reader was used to determine the a...

Watch this Scientific Journal Video about Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies at JoVE.com

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...

Precision cut lung slices method will preserve the structure and spatial orientations in three dimensions without regional bias and can serve as a live surrogate model for vessels and airway contractility measurements. In combination with time-lapse imaging, Western blot, and RNA analysis, precision cut lung slices contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases. To begin, place the euthanized adult mouse in a supine position and secure it in place by taping the paws and nose to the table with adhesive tape.Spray the ventral surface of the mouse with 70%ethanol. Use forceps to lift the abdominal skin of the mouse at the location of the bladder and surgical scissors to cut at the midline, moving superiorly to the cervical region to expose the trachea. Then use tweezers to lift the underlying fascia layer and use surgical scissors to expose the visceral organs and mediastinal compartment, again cutting inferiorly to superior.Cut the sternum at the midline to expose the heart and lung. Dissect the diaphragm carefully from the liver and the abdominal compartment. Locate the inferior vena cava under the intestines and cut it.After locating the right ventricle, use a 25 to 30 gauge butterfly needle to inject 0.5 milliliters of 1%fractionated heparin into the right ventricle and flush it slowly but steadily with 10 to 15 millimeters of PBS. After locating the larynx, use blunt tip tweezers to dissect the surrounding fascia and tissue, then place the surgical suture under the trachea and loosely tie a surgical knot. Place a small hole in the trachea superior to the string and cannulate with a 20 gauge blunt-ended needle.Tighten the suture around the needle and trachea using a surgical knot. Then inject 2.5 to 4 milliliters of agarose into the trachea and monitor for inflation of the lung. After the agarose injection, pour cold PBS over the lung to solidify the agarose.Then cut the trachea superior to the surgical suture and dissect the lung and heart from the mediastinum by removing any adhesions and fascia. After solidification, place the tissue in DMEM in a Petri dish on ice, and immediately proceed to slicing one lobe using a vibratome machine. To prepare the lung slices, attach the sample to the platform with superglue, keeping the medial side facing up.Then fill the sample container with PBS. Fill the container with ice to keep the surrounding PBS and sample cold. Turn on the vibratome.Adjust the thickness to 300 micrometers, frequency to 100 Hertz, amplitude to 0.6 millimeters, and speed to five micrometers per second. Using an Allen wrench, turn the blade holder into the safe position and open the jaws to insert a blade. Then tighten the jaws with an Allen wrench.Place the sample onto the box, ensuring it is submerged in PBS. Slide the box up in the correct position and make sure it is secure. Turn the blade holder into the appropriate position for slicing.Bring the vibratome blade up to the edge of the block and manually lower the blade until it is even with the top of the sample. Confirm the settings and run the vibratome. After the fresh slices are cut, remove the samples from the PBS and place them into a sterile Petri dish with 10 milliliters of DMEM containing one-time antimycotic antibiotic.After placing all the slices in a Petri dish, retract the blade entirely and use the Allen wrench to raise the blade into a safe position. Store the samples in DMEM at 37 degrees Celsius. And place a vibratome slice on a microscope slide.Use a cleaning wipe to remove excess medium. Place the sample on the phase contrast microscopy and use 10 to 20X magnification to identify a vessel. Then add 500 microliters of vasoconstrictors, such as 60 millimolar potassium chloride or one micromolar endothelin-1 to completely submerge the slide.Observe and capture the video by recording for 30 to 60 seconds. Before beginning the experiment, fill a small polystyrene foam box with one liter of liquid nitrogen and place two 1.5 milliliter microcentrifuge tubes in the container with liquid nitrogen. Place four to five fresh vibratome slices in a mortar bowl.Pour 5 to 10 milliliters of liquid nitrogen on top of the slices and quickly use the pestle to grind the slices into powder before the liquid nitrogen evaporates. Use a pre-chilled steel laboratory scooper to scoop the powder into the two chilled microcentrifuge tubes and lyse the powder by adding 500 microliters of RNA extraction reagent for RNA isolation or 500 microliters of RIPA buffer for protein lysis. In the representative viability experiment, the color change of the PCLS slices was detected from day zero to one and day nine to 10.The solution was initially blue and turned pink overnight, demonstrating viability. Color change typically occurred within one to four hours, but a longer time may be necessary. The daily difference between absorbance at 562 and 613 nanometer wavelengths for the vibratome-prepared tissue is shown here.The constriction of the vasculature in a vibratome-prepared lung tissue using endothelin-1 and potassium chloride can be visualized here. The preservation of tdTomato labeling in cells one week after tamoxifen injection is demonstrated in these lung slices. It is most important to perform the lung clearing and inflation step well to remove all blood from circulation before additional experiments and imaging.In addition to vascular diseases, PCLS can be applied to study physiobiology of airway-related disease, and can be further tested on human lung tissues in clinically relevant scenarios.

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Medicine

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the lung. Barometric plethysmography is a classic technique to evaluate the lung volume with a long history of clinical use. Volumetric capnography utilizes the profile of exhaled carbon dioxide to determine the volume of the conducting airways, or dead space, and provides an index of airways homogeneity. These techniques may be used independently, or in combination to evaluate the dependence of airways volume and homogeneity on lung volume. This paper provides detailed technical instructions to replicate these techniques and our representative data demonstrates that the airways volume and homogeneity are highly correlated to lung volume. We also provide a macro for the analysis of capnographic data, which can be modified or adapted to fit different experimental designs. The advantage of these measures is that their advantages and limitations are supported by decades of experimental data, and they can be made repeatedly in the same subject without expensive imaging equipment or technically advanced analysis algorithms. These methods may be particularly useful for investigators interested in perturbations that change both the functional residual capacity of the lung and airways volume.

Here, we describe two measures of pulmonary function &#8211; barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes.

Tools to measure lung and airways volume are critical for pulmonary researchers interested in evaluating the impact of disease or novel therapies on the ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...