Exploring alveolar recruitability using positive end-expiratory pressure in mice overexpressing TGF-ß1: a structure-function analysis.

Pre-injured lungs are prone to injury progression in response to mechanical ventilation. Heterogeneous ventilation due to (micro)atelectases imparts injurious strains on open alveoli (known as volutrauma). Hence, recruitment of (micro)atelectases by positive end-expiratory pressure (PEEP) is necessary to interrupt this vicious circle of injury but needs to be balanced against acinar overdistension. In this study, the lung-protective potential of alveolar recruitment was investigated and balanced against overdistension in pre-injured lungs. Mice, treated with empty vector (AdCl) or adenoviral active TGF-ß1 (AdTGF-ß1) were subjected to lung mechanical measurements during descending PEEP ventilation from 12 to 0 cmH2O. At each PEEP level, recruitability tests consisting of two recruitment maneuvers followed by repetitive forced oscillation perturbations to determine tissue elastance (H) and damping (G) were performed. Finally, lungs were fixed by vascular perfusion at end-expiratory airway opening pressures (Pao) of 20, 10, 5 and 2 cmH2O after a recruitment maneuver, and processed for design-based stereology to quantify derecruitment and distension. H and G were significantly elevated in AdTGF-ß1 compared to AdCl across PEEP levels. H was minimized at PEEP = 5-8 cmH2O and increased at lower and higher PEEP in both groups. These findings correlated with increasing septal wall folding (= derecruitment) and reduced density of alveolar number and surface area (= distension), respectively. In AdTGF-ß1 exposed mice, 27% of alveoli remained derecruited at Pao = 20 cmH2O. A further decrease in Pao down to 2 cmH2O showed derecruitment of an additional 1.1 million alveoli (48%), which was linked with an increase in alveolar size heterogeneity at Pao = 2-5 cmH2O. In AdCl, decreased Pao resulted in septal folding with virtually no alveolar collapse. In essence, in healthy mice alveoli do not derecruit at low PEEP ventilation. The potential of alveolar recruitability in AdTGF-ß1 exposed mice is high. H is optimized at PEEP 5-8 cmH2O. Lower PEEP folds and larger PEEP stretches septa which results in higher H and is more pronounced in AdTGF-ß1 than in AdCl. The increased alveolar size heterogeneity at Pao = 5 cmH2O argues for the use of PEEP = 8 cmH2O for lung protective mechanical ventilation in this animal model.

Microscopic and biochemical monitoring of endosomal trafficking and extracellular vesicle secretion in an endogenous in vivo model.

Extracellular vesicle (EV) secretion enables cell-cell communication in multicellular organisms. During development, EV secretion and the specific loading of signalling factors in EVs contributes to organ development and tissue differentiation. Here, we present an in vivo model to study EV secretion using the fat body and the haemolymph of the fruit fly, Drosophila melanogaster. The system makes use of tissue-specific EV labelling and is amenable to genetic modification by RNAi. This allows the unique combination of microscopic visualisation of EVs in different organs and quantitative biochemical purification to study how EVs are generated within the cells and which factors regulate their secretion in vivo. Characterisation of the system revealed that secretion of EVs from the fat body is mainly regulated by Rab11 and Rab35, highlighting the importance of recycling Rab GTPase family members for EV secretion. We furthermore discovered a so far unknown function of Rab14 along with the kinesin Klp98A in EV biogenesis and secretion.