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....

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

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 ....

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 the inn.......

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

List of materials used in this article

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