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

Background and aims: Idiopathic pulmonary fibrosis (IPF) is a fibrosing lung disease with un-known 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 quan-titative 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 alpha-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.