Impact of bronchoalveolar lavage procedures on pulmonary inflammation in nanoparticle inhalation models

Bronchoalveolar lavage fluid (BALF) analysis provides critical insights into pulmonary inflammation by identifying cellular components and inflammatory mediators in the airways and alveoli. However, variations in BALF collection protocols between laboratories limit cross-study comparison. Thus, optimal methodologies are necessary for future work on harmonization. In this study, we assessed critical parameters during BALF collection in rats by evaluating the number of lavages (1-5), the number of flushing repetitions (1 or 3 times), gentle lung massage, and the lavage volume adjusted for body weight. These procedures were evaluated under both non-inflamed, acutely inflamed, and chronically inflamed conditions induced by nanomaterials. Gentle lung massage, combined with three flushes (3FwM), resulted in the highest total cell recovery; however, it also increased variability and progressive cell depletion over multiple lavages. Conversely, performing a single flush without a massage (1FwoM) consistently produced stable and reproducible cytological and biochemical data across all inflammation models, thereby reducing experimental variability. In addition, both Dulbecco's phosphate-buffered saline (DPBS) and 0.9% saline were suitable lavage solutions, and DPBS supplemented with 10% fetal bovine serum effectively preserved BALF cell integrity for up to 1 week at 4°C. Automated cell counting offers superior reproducibility compared to manual counting, particularly under inflammatory conditions. Furthermore, we propose an equation to estimate the lavage volume applicable to the three rat strains. Thus, the proposed 1FwoM method represents a robust, standardized approach for collecting BALF, facilitating accurate comparisons, and enhancing regulatory inhalation toxicity assessments.