399 Establishing an in Vivo Model to Study Pulmonary Neutrophil Extracellular Trap (NET) Formation After Burn Injury

It is well known that burn patients suffer from serious lung complications such as acute respiratory distress syndrome, acute lung injury and pneumonia. However, the reasons for the development of these conditions after burn injury (BI) are not clearly understood. Several studies examined the importance of macrophages after burn injury. However, the involvement of neutrophils in lung complications after BI has not been studied in detail. Particularly, the importance of recently identified neutrophil extracellular traps (NETs) in the lungs after BI is unknown. NETs are considered to help trap infectious agents to protect the host; however, excess NETs could damage and destroy the airways and cause lung dysfunction. There are no good animal models available to study the pulmonary NETs during BI. Therefore, we established an in vivo model to study NETosis in the lungs after BI using an LPS model. We have first created a 15% body burn, and instilled various amounts of LPS (0–50 mg/kg) into the airways of C57B/6 mice. After various time points (3 h, 16 h, 24 h, 48 h, 72 h) we have collected bronchoalveolar lavage (BAL) fluid and blood samples. Immune cells present in the BAL fluid were deposited on slides by Cytospin preparations, stained and quantified by microscopy. Cell and platelet counts in the blood samples were determined by an automated cell counter, and confirmed by blood smears, H and E staining and microscopy. DNA-protein complexes present in the BAL supernatant were analyzed by agarose gel electrophoresis and pocigreen assays. Presence of a NET marker, citrullinated histone, was analyzed by Western blots. The data obtained from these studies show that neutrophils are not detectable in the airways under baseline or after BI; however, different numbers of neutrophils and amounts of NETs were present under various experimental conditions and time points-post BI. These data show that we could measure NET components in the airways of mice instilled with LPS after BI. Blood analyses show that cells concentrations also differ among various experimental conditions, indicating the importance of neutrophil and NET-promoting components (e.g., platelets) in the blood after BI and/or LPS instillation. We have successfully established a mouse model to study pulmonary NETosis in BI, and optimized the range of LPS concentrations and time points necessary to observe differences in NETosis under various experimental conditions. This model should help to understand the roles of NETs in pulmonary dysfunction after BI, and for testing potential drugs for correcting NET-mediated lung complications.