Investigating a mechanism through which lowering surface tension with sulforhodamine B improves oxygenation in an acute respiratory distress syndrome model
Introduction: We previously found that a non-toxic dye, sulforhodamine B (SRB), works with albumin present in alveolar edema liquid to lower surface tension, T, in injured lungs (Kharge, J Appl Physiol 118:355, 2015). Lowering T should improve oxygenation. Accordingly, we found, in an excessive tidal volume, VT, induced lung injury model, that intravenous (IV) SRB administration improved peripheral arterial oxygen saturation, SpO2 (Wu, J Appl Physiol 130:1305, 2021). However, mechanistic understanding of the T-oxygenation relation is incomplete. Here, we introduce a two-hit lung injury model that combines an inflammatory insult with excessive-VT ventilation. Under these conditions, we test SRB; measure SpO2; and begin investigating how low T improves oxygenation by quantifying edema liquid and observing septal capillary patency.
Materials and Methods: We handle animals according to a protocol approved by the Stevens Institute of Technology Institutional Animal Care and Use Committee. We anesthetize Wistar-Han rats (200-300 g, n=6), instill intratracheal LPS (0.75 mg/kg in 1 ml/kg saline) as a first injurious hit, give a tail vein injection of SRB (target 1 µM in plasma) or control saline and allow the rat to wake up. After 24 hr, we anesthetize the rat again and cannulate a tail vein for venous access. We cannulate the trachea and begin mechanical ventilation (Inspira, Harvard Apparatus). In an acclimation period, we provide 10 min of protective ventilation (6 ml/kg VT; 4 cmH2O positive end-expiratory pressure, PEEP; 75 breaths/minute, bpm; 1:2 inspiratory:expiratory ratio; 1.0 fraction of inspired oxygen, FiO2). As a second injurious hit, we provide 20 min of excessive VT ventilation (50 ml/kg; 0 cmH2O PEEP; 11 bpm). We then return to protective ventilation and reduce FiO2 to 0.21. Over the next 4 hrs, we monitor SpO2 and every time SpO2 drops below 90% we increase FiO2 by 0.2. At the end of the 4 hrs, we administer IV fluorescein (300 mg/kg) for subsequent imaging and sacrifice the rat with IV potassium chloride. We close a stopcock at the trachea and excise the lungs. We inflate the lungs to total lung capacity and deflate to 15 cmH2O. Using confocal microscopy (SP5, Leica Microsystems), we image subpleural alveoli. To quantify edema, we determine wet-to-dry ratio (W/D) of the lower right lobe and, in post analysis of confocal images, use imageJ to determine alveolar liquid thickness, tLLL (Fig. 1).
Results, Conclusions, and Discussions: During ventilation, it is necessary to increase FiO2 to 1.0 ± 0.0 in control groups but only to 0.5 ± 0.1 (p < 0.05) in SRB-treated groups). Additionally, SRB reduces W/D from 3.5 ± 0.0 to 2.1 ± 0.1 (p < 0.05). And SRB decreases aerated alveolar liquid lining layer thickness (Fig. 3).
Regardless of SRB administration, tLLL tends to be higher in aerated alveoli in flooded regions than in aerated alveoli in aerated regions (Fig. 3). Finally, we observe that SRB maintains alveolar capillary patency. Thus, we demonstrate that the efficacy of SRB extends to an inflammatory lung injury model. We show that the SRB-induced improvement in oxygenation correlates with decreased lung edema liquid and may be influenced by low-T-induced relief of capillary compression leading to increased perfusion. We note that SRB minimizes future injury but does not reverse existing injury, and that we administer SRB prophylactically. But having demonstrated SRB efficacy in the presence of inflammation, it should be possible to administer SRB early after onset of respiratory failure or diagnosis of acute respiratory distress syndrome, e.g. at the start of mechanical ventilation, and ameliorate subsequent disease progression. Further investigation is required to identify the time point up until which SRB can impact outcome.
Acknowledgements (Optional): Supported by R01 HL113577.