# High-resolution multimodal imaging reveals spatial and temporal heterogeneity of airway mucus plugging in mice with muco-obstructive lung disease

## TL;DR

This study uses advanced imaging to show how mucus builds up in the lungs of mice with a lung disease, revealing differences across ages and structures.

## Contribution

A novel multimodal imaging approach reveals spatiotemporal heterogeneity in mucus plugging across developmental stages in a mouse model of muco-obstructive lung disease.

## Key findings

- Mucus scores in βENaC-tg mice were significantly higher than in wild-type mice at both juvenile and adult stages.
- SRCT analysis showed a strong correlation between mucus area and contact ratios, indicating obstruction and adherence.
- SEM imaging revealed detailed ultrastructure of mucus plugs in juvenile βENaC-tg mice.

## Abstract

Mucus plugging is a hallmark of muco-obstructive lung diseases; however, the spatiotemporal dynamics of plug formation remain poorly understood. We used a multimodal high-resolution imaging approach to study mucus plugging in newborn (0–1 days), juvenile (13–16 days), and adult (49–59 days) βENaC-transgenic mice (βENaC-tg, n = 24), a model of muco-obstructive lung disease, in comparison to age-matched wild-type mice (n = 28). Micro-computed tomography (µCT) enabled whole lung mucus scoring on a scale from 0 (no obstruction) to 2 (> 50% obstruction) using an extended murine airway tree nomenclature. A subset of βENaC-tg lungs (5 juvenile, 5 adult) underwent localised synchrotron radiation-based computed tomography (SRCT) for segmentation-based calculation of mucus area and contact ratios as indicators of luminal obstruction and airway wall adherence. µCT-guided scanning electron microscopy (SEM) provided nanoscale visualisation of mucus plugs on selected βENaC-tg lung samples (3 juvenile). µCT revealed age-dependent, heterogeneous mucus plug formation, with significantly increased mucus scores in juvenile (0.45 ± 0.26) and adult (0.30 ± 0.17) βENaC-tg compared to wild-type mice (0.01 ± 0.02 with P < 0.01, and 0.01 ± 0.02 with P < 0.05, respectively). SRCT analysis showed a strong positive correlation between mucus area and contact ratios (r ≥ 0.80), while SEM revealed the mucus plug ultrastructure. This multimodal imaging approach highlights the spatiotemporal heterogeneity of mucus plugging, forming a basis for future research on targeted therapeutic strategies for muco-obstructive lung diseases.

The online version contains supplementary material available at 10.1038/s41598-025-22537-7.