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

摘要

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.

摘要

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.

引言

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....

方案

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 ....

结果

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.......

讨论

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.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
1 mL 注射器	BD	309626
15 mL 无菌离心管	Celltreat	229411
3 mL 注射器	BD	309585
50 mL 无菌离心管	Celltreat	229422
乙酰-β-乙酰甲胆碱	Millipore Sigma	62-51-1
抗生素-抗真菌	药 ThermoFisher	15240-062
CCD 相机	尼康	Nikon Ds-Ri2 相机
盖眼镜	Fisher Scientific	12-548-5CP; 12-548-5PP
低温小瓶	Fisher Scientific	430488
定制的激光扫描共聚焦显微镜			参考资料 18
DMEM/F12	Fisher Scientific	MT-10-092-CM
内皮素 1	Millipore Sigma	E7764
精细解剖剪刀	Fisher Scientific	NC9702861
冷冻容器	Sigma-Aldrich	C1562
猪皮明胶	Sigma-Aldrich	9000-70-8
汉克斯平衡盐溶液 （HBSS）	Thermo Fisher
止血钳	Fisher Scientific	16-100-117
	HEPES Thermo Fisher	15630080
高真空硅脂	Fisher Scientific	146355d
异丙醇	Sigma-Aldrich	W292907-1KG-K
金属垫圈	家得宝产品管理局	800442	Everbilt平垫圈 #10
显微解剖钳	Sigma-Aldrich	F4142
针头皮静脉套装 （25 G）	EXELINT	26708
NOC-5	开曼化学	16534
尼龙网组件	供应	U-CMN-300
俄勒冈绿 488 BAPTA-1 AM	Life Technologies	o-6807
相差显微镜	尼康	尼康 Eclipse TS 100
Pluronic F-127	Thermo Fisher	P-6867
剃须刀片	Personna		Personna 白色双刃剃须刀片包装 100 支
磺溴酞	Sigma-Aldrich	S0252
强力胶	Krazy 胶 Krazy 胶		，全
能 超纯低熔点琼脂糖	Thermo Fisher	16520050
振动切片机	精密	VF 310-0Z
振动切片机冷却块	精密	SKU-VM-CB12.5-NC
振动切片机标本管		精密 SKU VF-SPS-VM-12.5-NC
Y 形静脉导管	BD	383336	BD Saf-T-Intima 封闭式静脉导管