Elastic Fiber Profiling in Lung Tissue Using Quantitative Imaging Analyses

Summary

Here, we present a protocol detailing tissue preparation for elastic fiber visualization and quantitative profiling in murine lung tissue. This method enables robust assessment of tissue architecture, spatial fiber organization, and extracellular matrix remodeling in physiological and pathological conditions.

Abstract

Remodeling of the pulmonary extracellular matrix (ECM) is a key feature of lung injury and disease. Elastic fibers are a central component of the lung ECM, providing resilience and recoil necessary for normal respiratory function, and their disruption contributes to impaired tissue mechanics. This protocol describes a robust, reproducible workflow for the preparation and analysis of murine lung tissue, with a focus on the visualization and quantitative profiling of elastic fibers. It outlines optimized tissue processing and histological staining methods to preserve and detect elastin structures. In addition, the protocol details an image analysis pipeline enabling quantitative assessment of elastic fiber abundance, organization, and spatial distribution within the lung. The workflow further incorporates stain vector estimation in QuPath, color deconvolution in Fiji/ImageJ, and TWOMBLI-based image analysis to enable quantitative assessment of elastic fiber abundance, branching, and network complexity. Strategies for image acquisition, region-of-interest selection, parameter optimization, and quality control are included to ensure reproducibility and minimize user-dependent bias. Using this workflow, we successfully identified alterations in elastic fiber organization and network architecture in the lungs of obese mice, demonstrating its applicability for detecting disease-associated ECM remodeling. Together, these approaches provide a comprehensive framework to investigate elastic fiber remodeling and its contribution to tissue architecture in physiological and pathological conditions. This method facilitates the study of fibroblast-driven structural changes and supports mechanistic insights into ECM alterations underlying lung disease.

Introduction

Elastic fibers are essential components of the pulmonary extracellular matrix (ECM), composed primarily of elastin and associated microfibrillar proteins that assemble into highly organized, resilient networks¹. In the lung, elastic fibers are arranged along alveolar septa and peribronchial structures, where they provide the elasticity and recoil required for normal respiratory mechanics^1,2. As one of the most abundant structural ECM components in tissues such as the lung, aorta, and uterus, its integrity is critical for maintaining tissue compliance^1,....

Protocol

All mouse experiments described here were performed in accordance with the approval of the Animal Ethics Committee of the state of North Rhine-Westphalia, Germany (AZ: 81-02.04.2021.A221).

NOTE: Wear appropriate personal protective equipment (lab coat, gloves, eye protection) throughout the procedure.

1. Sampling and Preparation of murine lung tissue

NOTE: Mice were euthanized by isoflurane overdose according to institutional and governmental guidelines prior to tissue collection.

1. Carefully open the thoracic cavity with a midline incision, then cut the diap....

Results

This protocol provides quantitative parameters of the elastic fiber network across different lung regions and experimental conditions. TWOMBLI analysis, as previously described, yields parameters such as fiber length, branching density, lacunarity, and fractal dimension for extracellular matrix assessment¹⁰. Building upon this framework, our approach enables a comprehensive characterization of elastic fiber organization, allowing assessment of changes in fiber abundance, connectivity, uniformity, .......

Discussion

This protocol provides a robust, reproducible workflow for investigating murine pulmonary extracellular matrix (ECM) architecture, with a particular focus on fibroblast-driven elastic fiber remodeling. Elastic fibers are essential for maintaining lung compliance and recoil, and their disruption is a hallmark of multiple pulmonary diseases, including fibrosis, lung injury, aging, and metabolic disorders². Despite their central functional role, quantitative assessment of elastic fiber organization a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Axio CS2 Slide Scanner	Carl Zeiss
Coplin Glass Staining jar	Sigma-Aldrich	S6016
DPX Mountant	Sigma-Aldrich	6522
Ethanol (100%) for histology	Carl Roth	9065.4
Fiji		https://imagej.net/software/fiji/	version 1.54t
Formalin solution, neutral buffered 10%	Sigma	HT501128
HISTOSETTE I Tissue Processing/Embedding Cassettes	Sigma-Aldrich	H0542
microtome	Thermo Fischer Scietific	HM355S
Micrsocope Slides	VWR	631-0108
Mouse: C57BL/6J	The Jackson Laboratory	RRID: IMSR_JAX:000664	Strain #:000664
paraffin embedding system	Epredia HistoStar embedding module
Prism		https://www.graphpad.com	version 11
Qupath		https://qupath.github.io	version 0.7.0
Resorcin-Fuchsin	Waldeck	2.00E-30
TWOMBLI plugin		https://github.com/wershofe/TWOMBLI
Xylene	Applichem GmBH	14020172