Early detection of acute lung injury (ALI) remains challenging, as conventional diagnostics rarely capture initial molecular changes. We therefore examined whether extracellular vesicles (EVs) in bronchoalveolar lavage fluid (BALF) can detect early regional injury and distinguish initial stress mechanisms in a porcine ALI model. Unilateral ALI was induced using Triton X-100, followed by six hours of mechanical ventilation with either fixed positive end-expiratory pressure (PEEP) at 5 cmH(2)O or transpulmonary-pressure (TPP)-guided PEEP. Spatially separated BALF sampling allowed direct comparison between injured and contralateral mechanically stressed lungs. EV concentration and size distribution were quantified by nano-flow cytometry, while vesicular identity was confirmed by transmission electron microscopy and tetraspanin dot blot analyses. Furthermore, EV metrics were related to corresponding histologic injury scores. Across both PEEP strategies, EV concentration was markedly higher in both directly injured and contralateral mechanically stressed lungs compared with sham controls, indicating alveolar stress beyond the primary injury site. Median EV size differed significantly by injury type and correlated inversely with histologic injury, particularly with alveolar neutrophil infiltration. BALF-derived EV concentration and size may therefore provide complementary, spatially resolved molecular readouts of early ALI.
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