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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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 Prepare the working solution. 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…

Representative Results

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 …

Discussion

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…

Materials

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