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, 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.
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.
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
2. Inflation of mouse lungs with agarose and gelatin solution
3. Sectioning of lung lobes to thin slices
4. Analyzing contractile responses of intrapulmonary airways and arteries
5. Analyzing Ca2+ signaling of airway or vascular SMCs
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 the inner surface of the lumen. In contrast, the nearby pulmonary artery is featured by flat endothelium. When reaching the peripheral lung field, the conductive airways branch into respiratory ducts and sacs, surrounding the small intra-acinar arterioles (Figure 2C).
Utilizing mouse PCLS to assess the pulmonary airway and arterial contraction
Methacholine (MCh, 1 µM) induced airway contraction is demonstrated in Figure 2B. The airway contractile responses are quantified by the percentage of luminal area reduction following MCh exposure (Figure 2D). In contrast, the pulmonary artery presents no response to MCh stimuli (Figure 2B). Airways maintain similar dose-dependent contractile responses to MCh in the PCLS following 1-day or 5-day culture (Figure 2D). When the PCLS is exposed to endothelin (10 nM), both airways and pulmonary arteries constrict (Figure 2C,E), followed by NOC-5 (100 µM) induced relaxation (Figure 2E).
Utilizing mouse PCLS to assess the Ca2+ signaling of the airway and arterial SMC
The Ca2+ dye-loaded PCLS is observed under a confocal fluorescent microscope. The Ca2+ fluorescence in the airway (Figure 3B) and vascular (Figure 3C) SMCs is low at resting status, with no focal spark of intracellular Ca2+ signaling notable. Upon exposure to agonists, the Ca2+ fluorescence intensity elevates in the SMC (Figure 3B,C), usually from one spot and then propagating to the entire cell. The Ca2+ fluorescent waves repeatedly appear in the same cell as oscillatory signals (Figure 3D,E). In general, the frequency of Ca2+ oscillation increases as the agonist concentration rises until reaching a plateau level24. Airway SMC relaxation is associated with decreasing or cessation of Ca2+ oscillations25.
Figure 1: Orientation of mouse lung lobes for vibratome slicing. The mouse lung lobes are separated into individual ones for sections. (A) The left (1), right cranial (2), and caudal lobes (3) are trimmed near the hilum along the white dotted lines before sticking the flat cutting surface on the sample column. The placement of the left lobe is shown in (4). (B) The right middle lobe can be directly glued to the sample column. The right accessory lobe was not commonly used due to its small size. Appropriate orientation of different lobes ensures that most airways and pulmonary vessels present transverse sections in the PCLS. Scale bar = 1 cm. Please click here to view a larger version of this figure.
Figure 2: Contractile and relaxant responses of airways and pulmonary arteries in mouse PCLS. (A) Schematic showing the placement of a PCLS in a culture plate well for contraction assay. (B) Representative images showing an airway (black arrows) with a nearby pulmonary artery (black arrowheads) in HBSS at rest and following exposure to 1 µM methacholine (MCh). (C) Representative images showing an intra-acinar arteriole in HBSS at rest and upon exposure to 10 nM endothelin (Endo). (D) Dose-dependent airway contractile responses to MCh in PCLSs following 1-day (grey line) and 5-day (black dotted line) culture. Each point represents the average ± SEM of nine airways from two mice. (E) Representative images showing 10 nM endothelin-induced airway and pulmonary arterial contraction, followed by 100 µM NOC-5, a nitric oxide donor, induced relaxation. Scale bar = 100 μm. Please click here to view a larger version of this figure.
Figure 3: Ca2+ signaling of airway and pulmonary arterial SMCs in PCLS. (A) Schematic showing the setup of a chamber with a top and a bottom cover glass, grease seal, and a nylon mesh to hold a PCLS in the focal plane for Ca2+ imaging of SMCs. (B) Representative fluorescent images showing the Ca2+ signaling of airway SMCs at rest and following exposure to 1 µM MCh. Epi, epithelial cell. (C) Ca2+ fluorescent images of pulmonary arterial SMCs at rest and following exposure to 10 nM endothelin (Endo). The bold white arrows indicate the longitudinal axis of the pulmonary artery, and the dotted lines with end arrows indicate the helical distribution of vascular SMCs around the arterial wall. Scale bar = 20 µm. Oscillatory elevation of Ca2+ fluorescence intensity (Ft), in ratio to the fluorescence intensity at rest condition (F0), in an airway SMC to 1 µM MCh (D) and in a pulmonary arterial SMC in response to 10 nM endothelin stimulation (E). Please click here to view a larger version of this figure.
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 a water bath and using a heating lamp at the dissection table can be practiced to avoid quick agarose gelling. A quick injection could push more agarose to the lung parenchyma with higher compliance, hence needs to be avoided. The manual agarose injection usually takes around 5-7 s. Second, a necessary step at the end of agarose infilling is to push a small amount of air (~0.2 mL) to flush the agarose from the conductive airway to the distal alveoli space. Otherwise, the agarose would gel and stay in the lumen to resist the airway contraction. It is also worth noting that the agarose gelling inside the alveoli stays in place and never melts again at 37 °C in the incubator. The agarose gel plays an essential role in holding the 3D structure of lung tissue as in vivo maintained by the negative intrathoracic pressure. Third, perfusing pulmonary arteries with gelatin solution is essential to keep the arterial lumen open in the PCLS. The gelatin solution gels at room temperature as a mechanical blocker to resist vasoconstriction upon chemical and physical stimuli during the tissue section. Without gelatin inflation, the pulmonary arteries in lung slices usually collapse and detach from the surrounding interstitial tissue, even in the presence of high doses of mixed vasodilatory agents, including phentolamine, epinephrine, and nifedipine13,16. In contrast to an agarose gel, gelatin gel melts at 37 °C, flowing out of the vascular lumen after overnight incubation, leaving the arterial lumen free of obstruction prior to vascular reaction assay.
As the PCLS preserves pulmonary SMCs in situ and retains their contractile function in a nearly in vivo condition, it has been applied as a powerful platform to investigate the regulation of SMC contraction, especially the regulation via Ca2+ dependent mechanisms26. In particular, with a low-magnification multi-channel confocal or two-photon microscope, agonist-induced Ca2+ signaling in airway SMCs and associated luminal constriction can be captured simultaneously for mechanistic study13,20. Airway or vascular responsiveness measured using the PCLS method is expected to reflect cellular properties free of influences from the lung environment, such as the inflammatory milieu, and neural innervation of the response in the lung27,28. As such, the PLCS provides an experimental system to help distinguish intrinsic vs. secondary SMCs modification. In addition to investigating the contractile regulation of SMCs in health and disease models, PCLSs have been collected from different age groups to explore the functional adaptation of airway SMC during postnatal lung development and in response to environmental insults27. Moreover, PCLSs contain different-sized airways and blood vessels from the peripheral to the proximal lung field, enabling the investigation of a region-specific mechanism to regulate the pulmonary SMCs contractility in homeostasis and under pathogenic stimuli. Furthermore, as mouse PCLSs retain airway contractility in the culture medium for 7 days17, they have been used as an ex vivo model to examine or validate risk factors for SMC deregulation, such as cytokine or virus exposure29. Lastly, PCLS provides an ideal platform to screen vasodilatory or bronchodilatory medications. In particular, the bioassays using PCLS preparation are highly cost-effective, as one adult mouse can generate hundreds of lung slices. Using neighbor PCLSs in the control and treatment groups also significantly reduces the experimental bias from intergroup sample variation.
Considering the difference between rodent and human lung anatomy, the human PCLS is a more powerful tool for translational research. However, the limited availability of human lung tissue, especially diseased lung samples, remains a challenge. In contrast, mouse lung tissue, mouse models of human diseases, and transgenic mouse models are widely applied in biomedical and pharmacological research, making mouse PCLS an accessible and disease-relevant system13,30. In addition, the preservation of intrapulmonary arteries has been successful only in mouse PCLS preparation, which makes it a unique tool to explore the vascular deregulation in pulmonary vascular diseases such as pulmonary hypertension. Therefore, despite caveats associated with any disease models, a protocol for mouse PCLS preparation is invaluable to establish an ex vivo platform to investigate airway and pulmonary arteries in health and disease. We and others have reported lung research with human PCLSs31,32,33,34. In our experience, the protocol of human PLCS preparation is similar to the mouse one, except applying a higher agarose concentration of 2%, more agarose solution (3 L for one lung), and much larger-sized catheters to cannulate main,lobar, or segmental bronchi for agarose injection. Experience in mouse PLCS preparation helps tremendously with human lung slice preparation.
Despite numerous advantages of using PCLS preparation in pulmonary SMC research, it is crucial to be aware of the limitations of this technique. First, the PCLS remains a static system, lacking physiological breathing cycles that periodically stretch the lung parenchymal and airways. Neither does it contain blood circulation, which generates pulsive pressure on the vascular SMCs and shearing forces on endothelial cells. These mechanical variations in the PCLS could modify the contractile regulation of SMCs. Even though a full establishment of the air ventilation and blood circulation in PCLSs is unachievable, at least for the airway SMC study, vagarious efforts have been placed in the past decade to generate a "breathing" PCLS by stretching the tissue section with a variety of devices35,36,37. Second, pulmonary SMCs maintain their contractility only for a limited period. This time limit prevents the PLCS from modeling the subacute changes of SMCs, for instance, a process taking more than 6-7 days to modify airway SMCs. Since previous research reveals SMCs lose contractility due to a diminution of contractile proteins, optimizing the culture medium with an additional low dose of insulin has been shown to sustain the airway SMC contraction for up to 12 days17. Lastly, the genetic manipulation of pulmonary SMCs by plasmid or siRNA transfection of the PCLS remains unsuccessful. The technical barrier to transfect SMCs in the PCLS certainly warrants further investigation, as this method provides an alternative approach to the transgenic animal model for the mechanistic study of pulmonary SMCs. More importantly, this technique is the exclusive measure to achieve genetic modulation of human pulmonary SMCs in translational research.
This article provides a comprehensive description of preparing mouse PCLSs with well-preserved airways and pulmonary arteries and applying them in the contraction and Ca2+ signaling assays. PCLS preparation supports the smooth muscle functioning in a live lung environment while allowing mechanistic study access to cells in situ. This unique feature has made the PCLS preparation a versatile tool for studying the pulmonary airway and vascular SMCs in health and diseases.
The authors have nothing to disclose.
This work is supported by NIH grants, K08135443 (Y.B), 1R01HL132991 (X.A).
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 |