Hypercapnic acidosis impairs plasma membrane wound resealing in ventilator-injured lungs

Abstract

The objective of this study was to assess the effects of hypercapnic acidosis on lung cell injury and repair by confocal microscopy in a model of ventilator-induced lung injury. Three groups of normocapnic, hypocapnic, and hypercapnic rat lungs were perfused ex vivo, either during or after injurious ventilation, with a solution containing the membrane-impermeant label propidium iodide. In lungs labeled during injurious ventilation, propidium iodide fluorescence identifies all cells with plasma membrane wounds, both permanent and transient, whereas in lungs labeled after injurious ventilation propidium iodide fluorescence identifies only cells with permanent plasma membrane wounds. Hypercapnia minimized the adverse effects of high-volume ventilation on vascular barrier function, whereas hypocapnia had the opposite effect. Despite CO2-dependent differences in lung mechanics and edema the number of injured subpleural cells per alveolus was similar in the three groups (0.48 +/- 0.34 versus 0.51 +/- 0.19 versus 0.43 +/- 0.20 for hypocapnia, normocapnia, and hypercapnia, respectively). However, compared with normocapnia the probability of wound repair was significantly reduced in hypercapnic lungs (63 versus 38%; p < 0.02). This finding was subsequently confirmed in alveolar epithelial cell scratch models. The potential relevance of these observations for lung inflammation and remodeling after mechanical injury is discussed.

Figure 1.

Experimental design. For explanation, see text.

Figure 2.

Box plots of number of propidium iodide–positive cells per alveolus (PI/Alv) by treatment group with data points added. Fewer than 12 per group may appear because of duplicate values. Groups A and B refer to label timing with respect to injury (labeled during injurious ventilation, A; labeled after injurious ventilation, B). Mean PI/Alv is 0.25 lower for Group B than Group A for hypocapnia, 0.32 lower for normocapnia, and 0.16 lower for hypercapnia (overall, p = 0.39). The estimated difference between Group A and Group B is 0.07 lower for normocapnia than for hypocapnia (p = 0.58), 0.09 lower for hypocapnia than for hypercapnia (p = 0.49), and 0.15 lower for normocapnia than for hypercapnia (p = 0.082).

Figure 3.

Estimated resealing percentage by capnia group and 95% confidence intervals via a bootstrap resampling procedure. The estimates (and 95% confidence intervals) are 51% (23–71%) for hypocapnia, 63% (50–72%) for normocapnia, and 38% (16–53%) for hypercapnia. The estimated resealing percentage is 11% higher for normocapnia than for hypocapnia (p = 0.37), 13% higher for hypocapnia than for hypercapnia (p = 0.41), and 25% higher for normocapnia than for hypercapnia (p = 0.022).

Figure 4.

Plasma membrane–resealing rates after scratch injury under normocapnic (PCO₂ = 36.4; pH 7.47) hypocapnic (PCO₂ = 20; pH 7.66), and hypercapnic (PCO₂ = 119; pH 7.01) conditions. The fluorescence image insets demonstrate CO₂-dependent differences in the number of injured cells with transient (green) and permanent (red) plasma membrane wounds. The rate of plasma membrane repair in cells injured under normocapnic conditions was 73 ± 13% compared with only 60 ± 25 and 49 ± 20% in hypocapnic and hypercapnic preparations, respectively (*p < 0.05).