Abstract Rationale Murine models of lung injury are widely utilized to study inflammatory lung diseases. These models routinely employ analysis of bronchoalveolar lavage fluid (BALF), which is obtained as a terminal procedure on the day of harvest. Our group and others have reported the successful usage of a micro-bronchoscope (MB) to administer proteins, cells and bacteria to the tracheas and intrapulmonary airways of living mice 1,2,3. We hypothesize that this emerging technology could also enable serial BAL sampling during the course of inflammatory lung injury. To evaluate this, we assessed the feasibility of using a MB to obtain serial unilateral BALF in mice in an established model of acute lung injury (bleomycin). Methods Bleomycin (2 U/kg) or PBS (Control) was administered intratracheally to adult 8-12-week-old C57BL6J female mice (n = 5 per group) using standard technique. A MB was used to obtain BALF at 3, 5, and 10-days post injury (DPI) by instilling 200 ul of PBS into the left lobe under direct visualization. The percentage of fluid return was monitored and BALF was analyzed for total and differential cell counts and total protein concentration. Data analysis was performed in GraphPad Prism. Results As has been previously shown, over the course of the 10-day post-treatment follow-up period, mice treated with bleomycin lost ∼10% of their body weight compared to control animals whose weight remained stable (Figure 1A). The average BALF recovery was 91.2 ul (SD 33 ul) or 45.6% (SD 16%) of instilled volume and did not differ between the experimental groups or time points. As expected, the protein concentration (Figure 1B) and cellularity (Figure 1C) of the BALF increased in the bleomycin treated but not PBS treated animals. Conclusions We demonstrate for the first time, that a murine MB can be used to obtain serial BALF samples in the same animal. This work has broad implications as this technique could be used to enhance the study of the inflammatory process in many lung injury models, both enhancing scientific investigation and simultaneously decreasing the number of animals needed, a key ethical consideration in research. Future research is necessary to evaluate the feasibility of obtaining unilateral lavages to compare the inflammatory environment of an injured lung to an uninjured lung in the same animal. Funding This work was supported by CFF Grant 005666F223-CI (AK), Parker B. Francis Foundation Award (ANR), NIH-K23-HL173662 (ANR), NIH-R01HL172493 (MB/OE), and University of Pittsburgh Institutional Funds. This abstract is funded by: NIH, CFF, Parker B Francis Foundation
Kohn et al. (Fri,) studied this question.