Measuring Elastic Moduli Alterations in Oligomycin- and Blebbistatin-Treated Embroyic Chicken Lungs

Abstract

Avian and mammalian lungs share signaling pathways during development and both consist of epithelial tissue in the small airways and alveoli, embedded in mesenchyme. This thesis examines how either oligomycin or blebbistatin treatment affects the stiffness (elastic moduli) of embryonic day 5 chicken lung tissue. Oligomycin treatment was expected to increase elastic moduli, as the drug’s inhibition of oxidative phosphorylation can increase production of reactive oxygen species, promote the expression of extracellular matrix proteins, and cause local inflammation. In contrast, blebbistatin treatment was anticipated to decrease elastic moduli, because its NM II inhibition can weaken cell-cell adhesion and lead to incorrect mitotic spindle orientation during mitosis, increasing tissue permeability. Published experiments have also found that blebbistatin treatment reduced F-actin levels, hindered apical constriction, and prevented the emergence of new secondary bronchi. Combined nanoindentation results from multiple trials testing each treatment found no significant difference in stiffness between conditions. However, oligomycin-treated explants were significantly stiffer than control explants in two of four trials, while control explants were stiffer than blebbistatin-treated explants in one of three trials. As a supplement to stiffness measurements, immunofluorescence stained blebbistatin-treated explants had significantly lower mean levels of nuclei, F-actin, and fibronectin per focal plane, as well as significantly lower maximum levels of F-actin, E-cadherin, and fibronectin per focal plane compared to control explants. Additionally, image analysis before and after blebbistatin incubation revealed that significantly fewer secondary bronchi emerged in blebbistatin-treated explants than in control explants, aligning with previous experiments. This study can serve to enhance understanding of pulmonary compliance in disease states, such as stiffening in Acute Respiratory Distress Syndrome or excessive compliance in COPD. This understanding can be applied to the design and implementation of medical ventilation devices to reduce the risk of lung injury and lower the annual number of premature deaths—over 4 million as of 2024—in the United States linked to lung diseases.