Sulforhodamine B and exogenous surfactant effects on alveolar surface tension under acute respiratory distress syndrome conditions

Abstract

In the acute respiratory distress syndrome (ARDS), alveolar surface tension, T, may be elevated. Elevated T should increase ventilation-induced lung injury. Exogenous surfactant therapy, intended to lower T, has not reduced mortality. Sulforhodamine B (SRB) might, alternatively, be used to lower T. We test whether substances suspected of elevating T in ARDS raise T in the lungs and test the abilities of exogenous surfactant and SRB to reduce T. In isolated rat lungs, we micropuncture a surface alveolus and instill a solution of a purported T-raising substance: control saline, cell debris, secretory phospholipase A₂ (sPLA₂), acid, or mucins. We test each substance alone; with albumin, to model proteinaceous edema liquid; with albumin and exogenous surfactant; and with albumin and SRB. We determine T in situ in the lungs by combining servo-nulling pressure measurement with confocal microscopy and applying the Laplace relation. With control saline, albumin does not alter T, additional surfactant raises T, and additional SRB lowers T. The experimental substances, without or with albumin, raise T. Excepting under aspiration conditions, addition of surfactant or SRB lowers T. Exogenous surfactant activity is concentration and ventilation dependent. Sulforhodamine B, which could be delivered intravascularly, holds promise as an alternative therapeutic.NEW & NOTEWORTHY In the acute respiratory distress syndrome (ARDS), lowering surface tension, T, should reduce ventilation injury yet exogenous surfactant has not reduced mortality. We show with direct T determination in isolated lungs that substances suggested to elevate T in ARDS indeed raise T, and exogenous surfactant reduces T. Further, we extend our previous finding that sulforhodamine B (SRB) reduces T below normal in healthy lungs and show that SRB, too, reduces T under ARDS conditions.

Keywords: acute respiratory distress syndrome; sulforhodamine B; surface tension; surfactant therapy.

Fig. 1.

Protocol for obtaining cell debris. Methods for (1) homogenizing red blood cells (RBCs) to obtain whole cell debris or (2) lysing RBCs to obtain (2A) lysate supernatant, (2B) ghost solution, or (2C) lipid extract.

Fig. 2.

Solution effects on cells. Confocal images of alveoli after flooding with fluorescein (23 µM; green in images) in normal saline (A), 0.1 mg/mL secretory phospholipase A₂ (sPLA₂) IB (B), 0.01 N hydrochloric acid (HCl) (C), or 0.01 N HCl + 5% albumin (D). In A, airspace (labeled) and septa (dashed lines) are black. The latter indicates fluorescein exclusion by intact epithelium. In B–D, fluorescein appears to be concentrated in cells. Morphology suggests that the cell types may include macrophages (arrowheads, round), alveolar epithelial type I cells (open-headed arrows, thin and lining septum), and, likely, alveolar epithelial type II cells (closed-headed arrows, cuboidal), indicating damage to cell membranes and possible macrophage activation. Images (488-nm excitation, indicated laser power, 750 gain) taken ∼2 min after solution injection, at 15-μm subpleural depth. Due to saturation in (B) and (C) on left, lower-laser-power replicate images shown on right.

Fig. 3.

Solution effects on alveolar surface tension, T. Base flooding solutions are as follows: normal saline (control solution and solvent of experimental solutions); whole cell debris, with hemoglobin concentration of heparinized blood; secretory phospholipase A₂ (sPLA₂) IB, 0.1 mg/mL; sPLA₂ IIA, 2.5 ng/mL; HCl, 0.01 N; and porcine gastric mucin, 25 µg/mL. Additives are as shown. After ventilating twice between transpulmonary pressures, P_L, of 5 and 15 cmH₂O, lungs held at constant P_L of 15 cmH₂O during 10- to 15-min period required to determine T. For the Infasurf + ventilation group, we apply 50, rather than two, ventilation cycles between P_L of 5 and 15 cmH₂O. Further details provided in the text. Horizontal gray bar shows mean ± SD for T of normal liquid lining layer in aerated lungs, also at P_L of 15 cmH₂O, from (31). Data for “no additive” groups with saline, HCl, and mucins are from previous study (37). Statistics shown on figure; N.S. indicates not significant.

Fig. 4.

Red blood cell fraction effects on T. A: effects of 6×-lysate fractions, with additions as shown, on T. B: effects of all concentrations of lysate supernatant and solutions with matching concentrations of methemoglobin on T. Lysis water volume is multiple of original heparinized-blood volume. Surface tension determination, horizontal bar, and statistics as in Fig. 3.