Abstract Rationale Age is a major risk factor for lung disease. We sought to develop an ex vivo model of accelerated age-related lung disease to determine the effect of chemically induced aging on young and aged lungs. Hydrogen peroxide was used as an agent to induce oxidative stress-induced cellular senescence in precision-cut lung slices (PCLS). The aim was to determine whether PCLS could be used to study cellular senescence. Methods Mouse lungs were surgically extracted from mice. The trachea was catheterized and lungs were inflated with 15% gelatin, warmed to 37°. Inflated lungs were then cut with a vibratome (VF-300, Precisionary) to generate slices with a thickness of 300 microns. Precision-cut lung slices were cultured for 5 days in DMEM/F12 (GIBCO) with 0.1% fetal bovine serum under standard conditions. For senescence induction, slices were exposed to media with varying concentrations of hydrogen peroxide (1 or 10 μM) for 1 hour followed by a change to standard media. Following RNA extraction and quality control, qPCR and RNAseq were performed. Results Hydrogen peroxide-treated tissue exhibited alveolar simplification and reduced cell nucleus density, with more pronounced effects in aged lungs. Immunohistochemistry staining revealed a decrease in the proportion of Ki67 positive cells. There was a dose-dependent effect, with 10μM hydrogen peroxide-treated slices exhibiting the lowest proportion of Ki67-positive cells compared to control (0.9% versus 0.2%). The opposite pattern emerged for senescence marker p21, with the 10 μM dose being associated with the highest proportion of positive cells. We observed a larger relative increase in p21-positive cells in aged (10.6% versus 1.8%) compared to young PCLS (3.6% versus 0.1%). Expression of Cdkn1a (p21) by qPCR was increased in aged (p 0.05), but not in young (p = 0.34). In contrast, oxidative stress marker Nrf2 was robustly increased in a dose-dependent manner, with the strongest increase in the 10μM group in young (p 0.05) and aged PCLS (p 0.05). Differential gene expression of RNAseq data was performed to determine genes associated with hydrogen peroxide treatment (Figure). NADPH oxidases (Noxo1, Duoxa1, Duoxa2, Duox2) were prominently increased. Senescence markers (Gdf15, Trp53, and Il6) and SASP related genes (Ccl20, Il18rap, Cxcl5, Il11) also had increased expression. Proliferation markers such as Mki67 and Cdc25c had decreased expression following hydrogen peroxide treatment. Conclusions PCLS treated with hydrogen peroxide showed evidence of senescence induction that was higher in aged lungs. Our results demonstrate the value of PCLS as a robust model for studying aging and cellular senescence. This abstract is funded by: NHLBI
Man et al. (Fri,) studied this question.