This work is supported by NIH grants, K08135443 (Y.B), 1R01HL132991 (X.A).

All animal care was in accordance with the guidelines of the Institutional Animal Care and Use Committee of Massachusetts General Hospital. Wild-type C57/B6 male mice, 8 weeks of age, were used for the present study.
1. Experimental preparation
<ol>
	<li>Prepare the working solution.
	<ol>
		<li>Prepare 1x Hank&#39;s Balanced Salt Solution (HBSS, with Ca2+ and Mg2+, and pH balanced with 20 mM HEPES, see Table of Materials). Use the HBSS solution to prepare and process the PCLS. Keep HBSS solution cold on ice when preparing PCLSs.</li>
	</ol>
	</li>
	<li>Prepare the culture medium of PCLS by supplementing Dulbecco&#39;s Modified Eagle Medium and F-12 (DMEM-F12) with an antibiotic-antimycotic agent (1:100 volume ratio, see Table of Materials).</li>
	<li>Prepare 1.5% agarose and 6% gelatin solution.
	<ol>
		<li>Mix low melting point (LMP) agarose or gelatin powder (see Table of Materials) with HBSS solution separately in 15 mL sterile centrifuge tubes (or 50 mL tubes if solution volume &#62;10 mL) to final concentrations. 
		NOTE: Total volume of agarose solution = 5 mL/mouse x number of mice; gelatin solution = 2 mL/mouse x number of mice.</li>
		<li>Heat the solution tubes in boiling water until the powder completely dissolves. Keep both agarose and gelatin solutions in a 42 &#176;C water bath. 
		NOTE: A heating lamp at the dissection table can be useful to keep the operating environment warm and prevent agarose solution from solidifying before being injected into the mouse lung.</li>
	</ol>
	</li>
	<li>Submerge all dissection tools, including a pair of dissecting scissors, two pairs of curved micro-dissecting forceps, and a pair of hemostatic forceps in 70% ethanol solution for at least 20 min for sterilization.</li>
	<li>Keep a vibratome ready (see Table of Materials) to section the lung tissue into thin slices.</li>
	<li>Keep an inverted phase-contrast microscope with a CCD camera to evaluate the airway or vascular contractile responses and a custom-built laser scanning confocal microscope (see Table of Materials) to assess the Ca2+ signaling in pulmonary SMCs18.</li>
</ol>
2. Inflation of mouse lungs with agarose and gelatin solution
<ol>
	<li>Euthanize the mouse.
	<ol>
		<li>Place the mouse in a plastic chamber (around 750 cm3) with 5% isoflurane. Keep the mouse in the chamber until it stops breathing for at least 1 min. 
		NOTE: Other primary euthanasia methods, such as intraperitoneal injection of ketamine (240 mg/Kg) and xylazine (32 mg/Kg)19 or pentobarbital (0.3 mL)20, can be applied as alternatives to inhaled isoflurane.</li>
		<li>Place the mouse body on a dissection board in the supine position. Fix the body in position by pinning down the tail, front paws, and head with 25 G syringe needles, and sanitize the body with 70% ethanol spray.</li>
		<li>Cut open the mouse&#39;s abdomen with surgical scissors (incision, ~2 cm long x 2 cm wide). Then, cut off the abdominal aorta to deplete the blood volume.</li>
	</ol>
	</li>
	<li>Inflate lung lobes following the steps below.
	<ol>
		<li>Open the chest cavity carefully along the sternum and bilateral inferior rib cage above the diaphragm, and observe the lung lobes collapse when the chest cavity opens.</li>
		<li>Remove part of bilateral ventral rib cages to expose the heart. Avoid lung damage by pointing the sharp scissor tip away from the lung tissue.</li>
		<li>Use forceps to separate the thymus and soft tissue in the mouse neck to expose the trachea.</li>
		<li>Cut a small hole (1.2 mm in diameter) in the upper end of the trachea allowing passage of the tip of a 20 G Y-shaped IV catheter (see Table of Materials).</li>
		<li>Connect one adaptor port of the Y-shaped catheter with a 3 mL syringe prefilled with 0.5 mL of air, and the other port with a 3 mL syringe prefilled with 2 mL of warm 1.5% agarose solution (42 &#176;C).</li>
		<li>Inject agarose solution to fill the catheter and then push the catheter through the pre-cut opening into the trachea for 5-8 mm in length.</li>
		<li>Slowly inject the agarose solution at around 1 mL/5 s. The lung will expand along the proximal-to-distal axis. Stop injection when the edge of each lung lobe is inflated. 
		NOTE: Do not overinflate the lung, as this could rupture it. The volume of agarose solution to inflate the whole lung of a young adult mouse is about 1.3 &#177; 0.1 mL.</li>
		<li>Inject 0.2-0.3 mL of air from the other syringe to push the residual agarose in the catheter and conductive airways to the distal alveoli space.</li>
		<li>Clip close the trachea with a pair of curved hemostatic forceps (see Table of Materials).</li>
	</ol>
	</li>
	<li>Infill the pulmonary vasculature.
	<ol>
		<li>Fill a 1 mL syringe with warm 6% gelatin. Connect to a needle scalp vein catheter, infill the catheter with gelatin solution, and then puncture the right ventricle close to the inferior wall with the needle.</li>
		<li>Push the needle into the right ventricle for 2-3 mm in length and point the needle tip to the main pulmonary artery.</li>
		<li>Inject 0.2 mL of gelatin solution slowly into the right ventricle and pulmonary arterial vessels. 
		NOTE: The lung lobes appear slightly paler with appropriate gelatin inflation.</li>
		<li>Keep the needle in place for 5 min after injection, cool the lung lobes by pouring ice-cold HBSS solution on the heart and lung, then place the body with the dissection board in a 4 &#176;C refrigerator or on ice for a total of 10 min.</li>
		<li>Remove the mouse lung and heart from surrounding connective tissues with scissors. Then, separate each lung lobe and keep them in HBSS solution on ice.</li>
	</ol>
	</li>
</ol>
3. Sectioning of lung lobes to thin slices
<ol>
	<li>Trim and attach the lung lobe on the top of the specimen column with superglue, and orient the lobe (Figure 1A,B) to allow the cutting direction to be perpendicular to most airways from the hila to the lung surface.</li>
	<li>Use a vibratome with a fresh thin razor blade to cut the lung lobe into 150 &#181;m slices. Collect the slices in 100 mm sterile Petri dishes prefilled with cold HBSS solution. 
	NOTE: Set appropriate blade moving speed and oscillation frequency before starting slicing. A typical setting for a Compresstom is Speed level 4 and Oscillation level 4.</li>
	<li>Transfer slices to Petri dishes filled with DMEM/F-12 culture medium (20 slices/15 mL medium/dish) and maintain them in a 37 &#176;C incubator overnight before experiments. 
	NOTE: Mouse PCLSs can be maintained in culture for about 7 days without losing airway contractile responses17. The longevity of pulmonary arteries has not been thoroughly evaluated. In our observation, they retain intact structure and vasoreaction for at least 3 days in DMEM/F12 medium. For long-term storage, mouse PCLSs can be placed in cryovials filled with DMEM/F12 medium containing 10% DMSO (5 slices in 1 mL medium per vial), frozen in an isopropyl alcohol-filled container at -80 &#176;C overnight, and cryopreserved in liquid nitrogen for weeks to months21.</li>
</ol>
4. Analyzing contractile responses of intrapulmonary airways and arteries
<ol>
	<li>Place PCLSs in an HBSS-filled 24-well culture plate, one in each well. Locate the PCLS in the middle of the well, then remove the HBSS solution with a pipette.</li>
	<li>Find the target airway and vessel in the slice under the microscope, then cover the slice using a nylon mesh with a pre-cut central hole (2-3 mm in diameter) to expose the target area.</li>
	<li>Lay down a hollow metal washer on top of the mesh to hold the slices in place (Figure 2A).</li>
	<li>Add 600 &#956;L of HBSS solution to submerge the PCLS. Rest the slice for 10 min, then record the baseline images of airways or vessels.</li>
	<li>Induce the airway or vascular contraction by cautiously suctioning out the blank HBSS solution with a pipette and adding 600 &#956;L of HBSS with an agonist, such as 1 &#181;M of methacholine (MCh) or 10 nM of endothelin (see Table of Materials). 
	NOTE: KCl stimulation as a tool-set standard is not required prior to methacholine or endothelin exposure. 100 mM of KCl stimulation in mouse airways only induces a small and irregular airway contraction (10%-15%)20. Likewise, a priming methacholine stimulation is not obligated as we have confirmed that the same agonist, for instance, methacholine or serotonin, triggers similar airway contraction on the first and repetitive exposures20,22.</li>
	<li>Observe the reaction under the microscope until the luminal area change reaches an equilibratory status, and then record the airway or vascular images for analysis.</li>
	<li>Remove MCh or endothelin solution with a pipette and add a new 600 &#181;L HBSS solution with the same agonist concentration and a relaxant; observe and record the airway or vascular relaxation.</li>
	<li>Quantify the airway or vascular reaction following the steps below.
	<ol>
		<li>Load the images to NIH-sponsored free software FIJI.</li>
		<li>Select the Polygon Selection in the toolbar to outline the airway or vascular lumen on each frame.</li>
		<li>Choose Analyze &#62; Set Measurements &#62; Area.</li>
		<li>Choose Analyze &#62; Measure to quantify the area of interest. Results are shown in a separate window for statistical analysis.</li>
	</ol>
	</li>
</ol>
5. Analyzing Ca2+ signaling of airway or vascular SMCs
<ol>
	<li>Prepare the Ca2+ dye loading buffer.
	<ol>
		<li>Dissolve Ca2+ dye, Oregon Green 488 BAPTA-1-AM (see Table of Materials), 50 &#181;g (one vial) with 10 &#181;L of DMSO.</li>
		<li>Dissolve 0.2 g of Pluronic F-12 powder (see Table of Materials) in 1 mL of DMSO to generate a 20% Pluronic solution.</li>
		<li>Mix 10 &#181;L of 20% Pluronic solution with 10 &#181;L of Ca2+ dye-DMSO solution.</li>
		<li>Make Ca2+ dye loading buffer by adding 20 &#181;L of the mixture to 2 mL of HBSS solution with 200 &#181;M of sulfobromophthalein (see Table of Materials).</li>
	</ol>
	</li>
	<li>Place 15 mouse PCLSs in 2 mL of Ca2+ loading buffer and incubate at 30 &#176;C for 1 h. Then move the slices to 2 mL of HBSS solution with 100 &#181;M of sulfobromophthalein for an additional 30 min for fluorescent dye de-esterification at room temperature.</li>
	<li>Detect Ca2+ signaling of SMCs.
	<ol>
		<li>Place Ca2+ dye-loaded slices on a large cover glass.</li>
		<li>Fill a 3 mL syringe with high vacuum silicone grease. Squeeze the grease through an 18 G blunted needle to draw two parallel lines across the cover glass above and below the slice.</li>
		<li>Cover the slice using a nylon mesh between two grease lines. Place the second cover glass on top of the mesh to generate a chamber sealed by grease on the sides (Figure 3A). 
		NOTE: The nylon mesh is essential to keep the slice attached to the bottom coverslip and thus stay within the working distance of a high magnitude inverted objective, such as 40x oil.</li>
		<li>Add HBSS or agonist solution into the chamber from one end by pipetting manually or via a perfusion system. Remove the fluid from the chamber by suctioning from the other end with tissue paper or vacuum in the case of continuous fluid perfusion.</li>
		<li>Put the PCLS chamber on the microscope stage. Detect the Ca2+ signaling of SMC23 with a custom-built resonant scanning confocal microscope with a laser scan rate of 15 or 30 images/s. 
		NOTE: Alternatively, a high-speed commercial laser scanning confocal microscope, widely available at present, can be applied in the Ca2+ signaling assay of pulmonary SMCs in PCLS.</li>
	</ol>
	</li>
	<li>Quantify the Ca2+ fluorescence in SMCs.
	<ol>
		<li>Load the recorded image sequence to FIJI and choose the Image &#62; Type &#62; 8-bit grayscale.</li>
		<li>Select the Rectangle Selection in the toolbar and define a 5 pixel x 5 pixel region of interest (ROI) in a smooth muscle cell.</li>
		<li>Choose Analyze &#62; Set Measurements &#62; Mean gray value, representing the Ca2+ fluorescence intensity.</li>
		<li>If the position of an ROI changes with SMC contraction, choose Analyze &#62; Measure the Ca2+ fluorescent intensity frame-by-frame.</li>
		<li>If the ROI of SMC stays in a similar position in a stack of images, choose Image &#62; Stacks &#62; Measure stack of the Ca2+ fluorescent intensity. 
		NOTE: A custom-written macro can be plugged in to track the ROI within the SMC when it moves with contraction in an image stack and automatically measures the Ca2+ intensity (gray value) of ROI frame-by-frame.</li>
	</ol>
	</li>
</ol>

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<li>Liu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+precision+cut+lung+slices+as+a+translational+model+for+the+study+of+lung+biology%5BTitle%5D)+AND+%22Respiratory+Research%22%5BJournal%5D)">Use of precision cut lung slices as a translational model for the study of lung biology.</a> Respiratory Research. 20 (1), 162(2019).</li>
<li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+lung+tissue+slice+culture%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Mouse lung tissue slice culture.</a> Methods in Molecular Biology. 1940, 297-311 (2019).</li>
<li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD38+plays+an+age-related+role+in+cholinergic+deregulation+of+airway+smooth+muscle+contractility%5BTitle%5D)+AND+%22The+Journal+of+Allergy+and+Clinical+Immunology%22%5BJournal%5D)">CD38 plays an age-related role in cholinergic deregulation of airway smooth muscle contractility.</a> The Journal of Allergy and Clinical Immunology. 6749 (21), 01760-01767 (2021).</li>
<li>Khan, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+integrated+multiomic+and+quantitative+label-free+microscopy-based+approach+to+study+pro-fibrotic+signalling+in+ex+vivo+human+precision-cut+lung+slices%5BTitle%5D)+AND+%22The+European+Respiratory+Journal%22%5BJournal%5D)">An integrated multiomic and quantitative label-free microscopy-based approach to study pro-fibrotic signalling in ex vivo human precision-cut lung slices.</a> The European Respiratory Journal. 58 (1), (2021).</li>
<li>Kennedy, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+rhinovirus+39+infection+on+airway+hyperresponsiveness+to+carbachol+in+human+airways+precision+cut+lung+slices%5BTitle%5D)+AND+%22The+Journal+of+Allergy+and+Clinical+Immunology%22%5BJournal%5D)">Effects of rhinovirus 39 infection on airway hyperresponsiveness to carbachol in human airways precision cut lung slices.</a> The Journal of Allergy and Clinical Immunology. 141 (5), 1887-1890 (2018).</li>
<li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryopreserved+Human+precision-cut+lung+slices+as+a+bioassay+for+live+tissue+banking.+a+viability+study+of+bronchodilation+with+bitter-taste+receptor+agonists%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+Cell+and+Molecular+Biology%22%5BJournal%5D)">Cryopreserved Human precision-cut lung slices as a bioassay for live tissue banking. a viability study of bronchodilation with bitter-taste receptor agonists.</a> American Journal of Respiratory Cell and Molecular Biology. 54 (5), 656-663 (2016).</li>
<li>Mondoñedo, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+system+for+cyclic+stretching+of+precision-cut+lung+slices+during+acute+cigarette+smoke+extract+exposure%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">A high-throughput system for cyclic stretching of precision-cut lung slices during acute cigarette smoke extract exposure.</a> Frontiers in Physiology. 11, 566(2020).</li>
<li>Davidovich, N., Huang, J., Margulies, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reproducible+uniform+equibiaxial+stretch+of+precision-cut+lung+slices%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">Reproducible uniform equibiaxial stretch of precision-cut lung slices.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 304 (4), 210-220 (2013).</li>
<li>Ram-Mohan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+traction+microscopy+to+quantify+muscle+contraction+within+precision-cut+lung+slices%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">Tissue traction microscopy to quantify muscle contraction within precision-cut lung slices.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 318 (2), 323-330 (2020).</li>
</ol>

The authors have nothing to disclose.

The preparation of PCLS involves several critical steps. First, it is essential to inflate the lung lobe homogeneously to avoid the variation of tissue stiffness from uneven agarose distribution. As the liquid agarose rapidly gels in thin catheters or airways at a temperature below 37 &#176;C, the resultant filling defect in the distal lung field could increase the disparity of lung tissue stiffness and cause tissue tearing during the vibratome section. Therefore, keeping the low-melting agarose solution at 42 &#176;C in...

Smooth muscle cell (SMC) is a major structural cell type in the lung, primarily residing in the media wall of airways and pulmonary vessels. SMCs contract to alter the luminal caliber, thus regulating air and blood flow1,2. Therefore, contractile regulation of SMCs is essential to maintain the homeostasis of air ventilation and pulmonary circulation. In contrast, aberrant SMC contractility provokes obstructive airway or pulmonary vascular diseases like asthma and pulmonary arterial hypertension. However, the functional assessment of lung SMCs has been challenged by limited access to the lung tissue, especially those small airways and microvessels in the distal part of the lung2,3. Current solutions resort to indirect assays, such as measuring airflow resistance by Flexivent to reflect airway constriction, and checking pulmonary arterial blood pressure by right heart catheterization to assess pulmonary vasocontraction4,5. However, these indirect assays have multiple disadvantages, such as being confounded by structural factors, failing to capture the spatial diversity of airway or vascular responses in the whole lung scale6,7, and unfitting for the mechanistic study of contractile regulation at the cellular level. Therefore, alternative approaches using isolated primary cells, trachea/bronchi muscle strips8,9, or large vascular segments10 have been applied for the SMC study in vitro. Nevertheless, these methods also have limitations. For example, a quick phenotypical adaptation of primary SMCs in the culture condition11,12 makes it problematic to extrapolate findings from cell culture to in vivo settings. In addition, the contractile phenotype of SMCs in the isolated proximal airway or vascular segments may not represent the SMCs in the distal lung6,7. Moreover, the muscle force measurement at the tissue level remains dissociated from molecular and cellular events that are essential for mechanistic insight into contractile regulation.
Precision-cut lung slice (PCLS), a live lung tissue section, provides an ideal ex vivo tool to characterize pulmonary SMCs in a near in vivo microenvironment (i.e., preserved multi-cellular architecture and interaction)13. Since Drs. Placke and Fisher first introduced the preparation of lung slices from agarose-inflated rat and hamster lungs in the 1980s14,15, this technique has been advanced continuously to provide PCLSs with higher quality and greater versatility for biomedical research. One significant improvement is the enhancement of pulmonary arterial preservation by gelatin infusion in addition to lung inflation with agarose via the trachea. As a result, both the airway and pulmonary arteries are kept intact in the PCLS for ex vivo assessement16. Furthermore, the PCLS is viable for a prolonged time in culture. For instance, mouse PCLSs had no significant change in cell viability and metabolism for a minimum of 12 days in culture, as well as, they retained airway contractility for up to 7 days17. In addition, PCLS keeps different-sized airways or vessels for contraction and relaxation assays. Moreover, intracellular Ca2+ signaling of SMCs, the determinant factor of cell contractility, can be assayed with Ca2+ reporter dyes imaged by a confocal or 2-photon microscope13.
Considering the extensive application of the mouse model in lung research, a detailed protocol is described here for preparing mouse PCLS with intact airways and intrapulmonary arteries for ex vivo lung research. Using the prepared PCLSs, we subsequently demonstrated how to evaluate the airway and pulmonary arterial responses to constrictive or relaxant stimuli. In addition, the method of loading the PCLS with Ca2+ reporter dye and then imaging Ca2+ signaling of SMCs associated with contractile or relaxant responses are also described.

<table><tbody><tr><td>1 mL syringe</td><td>BD</td><td>309626</td><td></td></tr><tr><td>15 mL sterile centrifuge tubes</td><td>Celltreat</td><td>229411</td><td></td></tr><tr><td>3 mL syringe</td><td>BD</td><td>309585</td><td></td></tr><tr><td>50 mL sterile centrifuge tubes</td><td>Celltreat</td><td>229422</td><td></td></tr><tr><td>Acetyl-beta-methacholine</td><td>Millipore Sigma</td><td>62-51-1</td><td></td></tr><tr><td>Antibiotic-anitmycotic</td><td>Thermo Fisher</td><td>15240-062</td><td></td></tr><tr><td>CCD-camera</td><td>Nikon</td><td>Nikon Ds-Ri2 camera</td><td></td></tr><tr><td>Cover glassess</td><td>Fisher Scientific</td><td>12-548-5CP; 12-548-5PP</td><td></td></tr><tr><td>Cryogenic vials</td><td>Fisher Scientific</td><td>430488</td><td></td></tr><tr><td>Custom-built laser scanning confocal microscope</td><td></td><td></td><td>Details in Reference 18</td></tr><tr><td>DMEM/F12</td><td>Fisher Scientific</td><td>MT-10-092-CM</td><td></td></tr><tr><td>Endothelin 1</td><td>Millipore Sigma</td><td>E7764</td><td></td></tr><tr><td>Fine dissecting scissor</td><td>Fisher Scientific</td><td>NC9702861</td><td></td></tr><tr><td>Freezing container</td><td>Sigma-Aldrich</td><td>C1562</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma-Aldrich</td><td>9000-70-8</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Thermo Fisher</td><td>14025092</td><td></td></tr><tr><td>Hemostatic forcep</td><td>Fisher Scientific</td><td>16-100-117</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher</td><td>15630080</td><td></td></tr><tr><td>High vaccum silicone grease</td><td>Fisher Scientific</td><td>146355d</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Sigma-Aldrich</td><td>W292907-1KG-K</td><td></td></tr><tr><td>Metal washers</td><td>Home Depot Product Authority</td><td>800442</td><td>Everbilt Flat Washers #10</td></tr><tr><td>Micro-dissecting forcep</td><td>Sigma-Aldrich</td><td>F4142</td><td></td></tr><tr><td>Needle scalp vein set (25 G)</td><td>EXELINT</td><td>26708</td><td></td></tr><tr><td>NOC-5</td><td>Cayman Chemical</td><td>16534</td><td></td></tr><tr><td>Nylon mesh</td><td>Component Supply</td><td>U-CMN-300</td><td></td></tr><tr><td>Oregon green 488 BAPTA-1 AM</td><td>Life Technologies</td><td>o-6807</td><td></td></tr><tr><td>Phase-contrast microscope</td><td>Nikon</td><td>Nikon Eclipse TS 100</td><td></td></tr><tr><td>Pluronic F-127</td><td>Thermo Fisher</td><td>P-6867</td><td></td></tr><tr><td>Razor blades</td><td>Personna</td><td></td><td>Personna Double Edge Razor Blades in White Wrapper 100 count</td></tr><tr><td>Sulfobromophthalein</td><td>Sigma-Aldrich</td><td>S0252</td><td></td></tr><tr><td>Superglue</td><td>Krazy Glue</td><td></td><td>Krazy Glue, All purpose</td></tr><tr><td>Ultrapure low melting point agarose</td><td>Thermo Fisher</td><td>16520050</td><td></td></tr><tr><td>Vibratome</td><td>Precisionary</td><td>VF 310-0Z</td><td></td></tr><tr><td>Vibratome chilling block</td><td>Precisionary</td><td>SKU-VM-CB12.5-NC</td><td></td></tr><tr><td>Vibratome specimen tube</td><td>Precisionary</td><td>SKU VF-SPS-VM-12.5-NC</td><td></td></tr><tr><td>Y shaped IV catheter</td><td>BD</td><td>383336</td><td>BD Saf-T-Intima closed IV catheter</td></tr></tbody></table>

utilizing the precision-cut lung slice to study the contractile regulation of airway and intrapulmonary arterial smooth muscle

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, respectively, hence playing a critical role in the homeostasis of the pulmonary system. Deregulation of SMC contractility contributes to several pulmonary diseases, including asthma and pulmonary hypertension. However, due to limited tissue access and a lack of culture systems to maintain in vivo SMC phenotypes, molecular mechanisms underlying the deregulated SMC contractility in these diseases remain fully identified. The precision-cut lung slice (PCLS) offers an ex vivo model that circumvents these technical difficulties. As a live, thin lung tissue section, the PCLS retains SMC in natural surroundings and allows in situ tracking of SMC contraction and intracellular Ca2+ signaling that regulates SMC contractility. Here, a detailed mouse PCLS preparation protocol is provided, which preserves intact airways and intrapulmonary arteries. This protocol involves two essential steps before subjecting the lung lobe to slicing: inflating the airway with low-melting-point agarose through the trachea and infilling pulmonary vessels with gelatin through the right ventricle. The PCLS prepared using this protocol can be used for bioassays to evaluate Ca2+-mediated contractile regulation of SMC in both the airway and the intrapulmonary arterial compartments. When applied to mouse models of respiratory diseases, this protocol enables the functional investigation of SMC, thereby providing insight into the underlying mechanism of SMC contractility deregulation in diseases.

Mouse PCLS preparation preserving intact intrapulmonary airways and arteries 
A 150 &#181;m thick PCLS was observed under the inverted phase-contrast microscope. In mouse lungs, conductive airways are accompanied by intrapulmonary arteries, running from the hilus to the peripheral lung. A representative pulmonary airway-artery bundle in a mouse PCLS is shown in Figure 2B. The airway can be easily identified by cuboidal epithelial cells with active cilial beating lining ...

Watch this Scientific Journal Video about Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle at JoVE.com

Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle

The present protocol describes preparing and utilizing mouse precision-cut lung slices to assess the airway and intrapulmonary arterial smooth muscle contractility in a nearly in vivo milieu.

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, ...

utilizing-precision-cut-lung-slice-to-study-contractile-regulation

Research

JoVE Journal

Biology

3.4K Views.  Massachusetts General Hospital and Harvard Medical School. The present protocol describes preparing and utilizing mouse precision-cut lung slices to assess the airway and intrapulmonary arterial smooth muscle contractility in a nearly in vivo milieu.

Video: Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

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 ...

Medicine

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Immunology and Infection

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

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.

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.

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

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

Innovative Lung Imaging Using Agarose-Embedded Vibratome Sections

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

Author Spotlight: Studying Spatial Protein Expression Using Agarose Embedded Lung Tissue Sections 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...

Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle

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Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging