Yang Xi, Huang Xue-Jun, Chen Zhang, Xu Ai-Li, Zhou Hua, Bi Xiao-Li, Yan Pei-Yu, Xie Ying
State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Macau, China.
Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine, Guangzhou, Guangdong Province, China.
Heliyon. 2023 Feb 10;9(3):e13598. doi: 10.1016/j.heliyon.2023.e13598. eCollection 2023 Mar.
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.
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.
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 (### 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.
Provided a quantifying method for Micro-CT images in an optimal and repeatable pulmonary fibrosis mice model for exploring novel therapeutic interventions.
特发性肺纤维化（IPF）是一种病因不明的纤维化性肺病，可导致咳嗽和呼吸困难，也是影响新冠病毒疾病幸存者生活质量的最常见后遗症之一。IPF患者无法治愈。我们旨在开发一种基于微型计算机断层扫描（micro-CT）图像进行纤维化定量的可靠IPF动物模型，用于新药研发，因为文献报道了不同的博来霉素给药途径、剂量和间隔时间，且在动物研究中尚无基于micro-CT图像对肺纤维化进行定量评估的方法。
我们通过研究生存率、肺组织病理学、micro-CT、外周血CD4和CD8细胞以及细胞因子，比较了C57BL/6小鼠气管内给予三种剂量（1.25mg/kg、2.5mg/kg和5mg/kg）博来霉素及实验间隔时间（14天和21天）的情况。此外，开发了一种简单可靠的新方法，通过使用Image J软件基于Micro-CT图像对活体小鼠的纤维化进行评分，该软件将肺部Micro-CT图像中的暗区转换为黑色背景上的亮色。
小鼠肺中羟脯氨酸、炎症细胞因子、纤维化病理变化和胶原沉积水平呈博来霉素剂量依赖性和时间依赖性，体重减轻情况也如此。基于上述结果，给予1.25mg/kg博来霉素后21天的小鼠模型具有最佳的肺纤维化，生存率高且毒性低。博来霉素处理组小鼠的亮区（灰度值为9.86±0.72）显著降低，表明与正常组相比，博来霉素损伤小鼠的肺泡气腔面积显著减小（###0.001），而给予吡非尼酮后亮区（灰度值）增加至21.71±2.95，接近正常小鼠观察到的值（灰度值为23.23±1.66），这与Col1A1和α-SMA的蛋白水平一致。值得注意的是，每组连续六幅图像的标准差表明了这种开发的定量方法对每只小鼠第五肋骨处拍摄的micro-CT图像的准确性。
为探索新型治疗干预措施，在最佳且可重复的肺纤维化小鼠模型中提供了一种对Micro-CT图像进行定量的方法。
