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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 flow^1,2. 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 lung^2,3. 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 vasocontraction^4,5. 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 scale^6,7, and unfitting for the mechanistic study of contractile regulation at the cellular level. Therefore, alternative approaches using isolated primary cells, trachea/bronchi muscle strips^8,9, 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 condition^11,12 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 lung^6,7. 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 1980s^14,15, 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.

Protokół

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

1. Prepare the working solution.
   1. Prepare 1x Hank'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.
2. Prepare the culture medium of PCLS by supplementing Dulbecco's Modified Eagle Medium and F-12 (DMEM-F12) with an antibiotic-antimycotic agent (1:100 volume ratio, see Table of Materials).
3. Prepare 1.5% agarose and 6% gelatin solution.
   1. 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 >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.
   2. Heat the solution tubes in boiling water until the powder completely dissolves. Keep both agarose and gelatin solutions in a 42 °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.
4. 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.
5. Keep a vibratome ready (see Table of Materials) to section the lung tissue into thin slices.
6. 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.

2. Inflation of mouse lungs with agarose and gelatin solution

1. Euthanize the mouse.
   1. 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.
   2. 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.
   3. Cut open the mouse's abdomen with surgical scissors (incision, ~2 cm long x 2 cm wide). Then, cut off the abdominal aorta to deplete the blood volume.
2. Inflate lung lobes following the steps below.
   1. 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.
   2. 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.
   3. Use forceps to separate the thymus and soft tissue in the mouse neck to expose the trachea.
   4. 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).
   5. 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 °C).
   6. 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.
   7. 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 ± 0.1 mL.
   8. 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.
   9. Clip close the trachea with a pair of curved hemostatic forceps (see Table of Materials).
3. Infill the pulmonary vasculature.
   1. 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.
   2. Push the needle into the right ventricle for 2-3 mm in length and point the needle tip to the main pulmonary artery.
   3. 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.
   4. 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 °C refrigerator or on ice for a total of 10 min.
   5. 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.

3. Sectioning of lung lobes to thin slices

1. 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.
2. Use a vibratome with a fresh thin razor blade to cut the lung lobe into 150 µm 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.
3. Transfer slices to Petri dishes filled with DMEM/F-12 culture medium (20 slices/15 mL medium/dish) and maintain them in a 37 °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 °C overnight, and cryopreserved in liquid nitrogen for weeks to months²¹.

4. Analyzing contractile responses of intrapulmonary airways and arteries

1. 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.
2. 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.
3. Lay down a hollow metal washer on top of the mesh to hold the slices in place (Figure 2A).
4. Add 600 μL of HBSS solution to submerge the PCLS. Rest the slice for 10 min, then record the baseline images of airways or vessels.
5. Induce the airway or vascular contraction by cautiously suctioning out the blank HBSS solution with a pipette and adding 600 μL of HBSS with an agonist, such as 1 µM 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 exposures^20,22.
6. 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.
7. Remove MCh or endothelin solution with a pipette and add a new 600 µL HBSS solution with the same agonist concentration and a relaxant; observe and record the airway or vascular relaxation.
8. Quantify the airway or vascular reaction following the steps below.
   1. Load the images to NIH-sponsored free software FIJI.
   2. Select the Polygon Selection in the toolbar to outline the airway or vascular lumen on each frame.
   3. Choose Analyze > Set Measurements > Area.
   4. Choose Analyze > Measure to quantify the area of interest. Results are shown in a separate window for statistical analysis.

5. Analyzing Ca²⁺ signaling of airway or vascular SMCs

1. Prepare the Ca²⁺ dye loading buffer.
   1. Dissolve Ca²⁺ dye, Oregon Green 488 BAPTA-1-AM (see Table of Materials), 50 µg (one vial) with 10 µL of DMSO.
   2. Dissolve 0.2 g of Pluronic F-12 powder (see Table of Materials) in 1 mL of DMSO to generate a 20% Pluronic solution.
   3. Mix 10 µL of 20% Pluronic solution with 10 µL of Ca²⁺ dye-DMSO solution.
   4. Make Ca²⁺ dye loading buffer by adding 20 µL of the mixture to 2 mL of HBSS solution with 200 µM of sulfobromophthalein (see Table of Materials).
2. Place 15 mouse PCLSs in 2 mL of Ca²⁺ loading buffer and incubate at 30 °C for 1 h. Then move the slices to 2 mL of HBSS solution with 100 µM of sulfobromophthalein for an additional 30 min for fluorescent dye de-esterification at room temperature.
3. Detect Ca²⁺ signaling of SMCs.
   1. Place Ca²⁺ dye-loaded slices on a large cover glass.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
4. Quantify the Ca²⁺ fluorescence in SMCs.
   1. Load the recorded image sequence to FIJI and choose the Image > Type > 8-bit grayscale.
   2. Select the Rectangle Selection in the toolbar and define a 5 pixel x 5 pixel region of interest (ROI) in a smooth muscle cell.
   3. Choose Analyze > Set Measurements > Mean gray value, representing the Ca²⁺ fluorescence intensity.
   4. If the position of an ROI changes with SMC contraction, choose Analyze > Measure the Ca²⁺ fluorescent intensity frame-by-frame.
   5. If the ROI of SMC stays in a similar position in a stack of images, choose Image > Stacks > 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.

Wyniki

Mouse PCLS preparation preserving intact intrapulmonary airways and arteries
A 150 µm 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 ...

Dyskusje

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 °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 °C in...

Materiały

Name	Company	Catalog Number	Comments
1 mL syringe	BD	309626
15 mL sterile centrifuge tubes	Celltreat	229411
3 mL syringe	BD	309585
50 mL sterile centrifuge tubes	Celltreat	229422
Acetyl-beta-methacholine	Millipore Sigma	62-51-1
Antibiotic-anitmycotic	Thermo Fisher	15240-062
CCD-camera	Nikon	Nikon Ds-Ri2 camera
Cover glassess	Fisher Scientific	12-548-5CP; 12-548-5PP
Cryogenic vials	Fisher Scientific	430488
Custom-built laser scanning confocal microscope			Details in Reference 18
DMEM/F12	Fisher Scientific	MT-10-092-CM
Endothelin 1	Millipore Sigma	E7764
Fine dissecting scissor	Fisher Scientific	NC9702861
Freezing container	Sigma-Aldrich	C1562
Gelatin from porcine skin	Sigma-Aldrich	9000-70-8
Hanks' Balanced Salt Solution (HBSS)	Thermo Fisher	14025092
Hemostatic forcep	Fisher Scientific	16-100-117
HEPES	Thermo Fisher	15630080
High vaccum silicone grease	Fisher Scientific	146355d
Isopropyl alcohol	Sigma-Aldrich	W292907-1KG-K
Metal washers	Home Depot Product Authority	800442	Everbilt Flat Washers #10
Micro-dissecting forcep	Sigma-Aldrich	F4142
Needle scalp vein set (25 G)	EXELINT	26708
NOC-5	Cayman Chemical	16534
Nylon mesh	Component Supply	U-CMN-300
Oregon green 488 BAPTA-1 AM	Life Technologies	o-6807
Phase-contrast microscope	Nikon	Nikon Eclipse TS 100
Pluronic F-127	Thermo Fisher	P-6867
Razor blades	Personna		Personna Double Edge Razor Blades in White Wrapper 100 count
Sulfobromophthalein	Sigma-Aldrich	S0252
Superglue	Krazy Glue		Krazy Glue, All purpose
Ultrapure low melting point agarose	Thermo Fisher	16520050
Vibratome	Precisionary	VF 310-0Z
Vibratome chilling block	Precisionary	SKU-VM-CB12.5-NC
Vibratome specimen tube	Precisionary	SKU VF-SPS-VM-12.5-NC
Y shaped IV catheter	BD	383336	BD Saf-T-Intima closed IV catheter