A novel quantification method of lung fibrosis based on Micro-CT images developed with the optimized pulmonary fibrosis mice model induced by bleomycin

Xi Yang; Xue-Jun Huang; Zhang Chen; Ai-Li Xu; Hua Zhou; Xiao-Li Bi; Pei-Yu Yan; Ying Xie

doi:10.1016/j.heliyon.2023.e13598

A novel quantification method of lung fibrosis based on Micro-CT images developed with the optimized pulmonary fibrosis mice model induced by bleomycin

Heliyon. 2023 Feb 10;9(3):e13598. doi: 10.1016/j.heliyon.2023.e13598. eCollection 2023 Mar.

Authors

Xi Yang¹, Xue-Jun Huang^{2

3}, Zhang Chen¹, Ai-Li Xu^{2

3}, Hua Zhou⁴, Xiao-Li Bi^{2

3}, Pei-Yu Yan², Ying Xie⁴

Affiliations

¹ State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Macau, China.
² Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine, Guangzhou, Guangdong Province, China.
³ Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine, Guangzhou, Guangdong Province, China.
⁴ State Key Laboratory of Dampness Syndrome of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China.

Abstract

Background and aims: Idiopathic pulmonary fibrosis (IPF) is a fibrosing lung disease with unknown etiology, leading to cough and dyspnoea, which is also one of the most common sequelae affecting the quality of life of COVID-19 survivors. There is no cure for IPF patients. We aim to develop a reliable IPF animal model with quantification of fibrosis based on Micro-Computer Tomography (micro-CT) images for the new drug discovery, because different bleomycin administration routes, doses, and intervals are reported in the literature, and there is no quantitative assessment of pulmonary fibrosis based on micro-CT images in animal studies.

Methods: We compared three dosages (1.25 mg/kg, 2.5 mg/kg, and 5 mg/kg) of intratracheal bleomycin administration and experiment intervals (14 and 21 days) in C57BL/6 mice by investigating survival rates, pulmonary histopathology, micro-CT, peripheral CD4⁺ & CD8⁺ cells, and cytokines. Moreover, a simple and reliable new method was developed for scoring fibrosis in live mice based on Micro-CT images by using Image J software, which transfers the dark sections in pulmonary Micro-CT images to light colors on a black background.

Results: The levels of hydroxyproline, inflammation cytokine, fibrotic pathological changes, and collagen deposition in the lungs of mice were bleomycin dose-dependent and time-dependent as well as the body weight loss. Based on the above results, the mice model at 21 days after being given bleomycin at 1.25 mg/kg has optimal pulmonary fibrosis with a high survival rate and low toxicity. There is a significant decrease in the light area (gray value at 9.86 ± 0.72) in the BLM mice, indicating that a significant decrease in the alveolar air area was observed in BLM injured mice compared to normal groups (###p < 0.001), while the Pirfenidone administration increased the light area (gray value) to 21.71 ± 2.95 which is close to the value observed in the normal mice (gray value at 23.23 ± 1.66), which is consistent with the protein levels of Col1A1, and α-SMA. Notably, the standard deviations for the consecutive six images of each group indicate the precision of this developed quantitation method for the micro-CT image taken at the fifth rib of each mouse.

Conclusion: Provided a quantifying method for Micro-CT images in an optimal and repeatable pulmonary fibrosis mice model for exploring novel therapeutic interventions.

Keywords: Bleomycin-induced pulmonary fibrosis mice model; Dosages; Experiment intervals; Quantitative micro-CT assessment.