Mechanical ventilation-induced alterations of intracellular surfactant pool and blood-gas barrier in healthy and pre-injured lungs

Abstract

Mechanical ventilation triggers the manifestation of lung injury and pre-injured lungs are more susceptible. Ventilation-induced abnormalities of alveolar surfactant are involved in injury progression. The effects of mechanical ventilation on the surfactant system might be different in healthy compared to pre-injured lungs. In the present study, we investigated the effects of different positive end-expiratory pressure (PEEP) ventilations on the structure of the blood-gas barrier, the ultrastructure of alveolar epithelial type II (AE2) cells and the intracellular surfactant pool (= lamellar bodies, LB). Rats were randomized into bleomycin-pre-injured or healthy control groups. One day later, rats were either not ventilated, or ventilated with PEEP = 1 or 5 cmH₂O and a tidal volume of 10 ml/kg bodyweight for 3 h. Left lungs were subjected to design-based stereology, right lungs to measurements of surfactant proteins (SP-) B and C expression. In pre-injured lungs without ventilation, the expression of SP-C was reduced by bleomycin; while, there were fewer and larger LB compared to healthy lungs. PEEP = 1 cmH₂O ventilation of bleomycin-injured lungs was linked with the thickest blood-gas barrier due to increased septal interstitial volumes. In healthy lungs, increasing PEEP levels reduced mean AE2 cell size and volume of LB per AE2 cell; while in pre-injured lungs, volumes of AE2 cells and LB per cell remained stable across PEEPs. Instead, in pre-injured lungs, increasing PEEP levels increased the number and decreased the mean size of LB. In conclusion, mechanical ventilation-induced alterations in LB ultrastructure differ between healthy and pre-injured lungs. PEEP = 1 cmH₂O but not PEEP = 5 cmH₂O ventilation aggravated septal interstitial abnormalities after bleomycin challenge.

Keywords: Alveolar epithelial type II cells; Blood–gas barrier; Lamellar bodies; Mechanical ventilation; Stereology; Surfactant.

Fig. 1

Ultrastructure of the blood–gas barrier. In healthy controls (H) the ultrastructural presentation of the blood–gas barrier consisting of the alveolar epithelium (AE), the interstitium and the endothelium (endo) was unaffected by ventilation. After bleomycin challenge (B) however, signs of injury of the alveolar epithelial (AE) cells could be found. AE cells were either swollen (characterized by cytoplasmic clearance) or completely missing so that the basal lamina (bl) was denuded. In healthy controls, the interstitium, consisting of extracellular matrix (ECM) and interstitial cells (IC) is thin and compact; while after bleomycin challenge, there is widening in some areas. Further abbreviations: col collagen fibrils, air alveolar airspace, caplumen capillary lumen, ery erythrocyte. Scale bar: 1 µm

Fig. 2

Stereological data characterizing abnormalities of the blood–gas barrier. Bleomycin challenge increases the absolute volume of the interstitium within the interalveolar septa (a), in particular after mechanical ventilation with PEEP = 1 cmH₂O. The increase in V(inter,sep) is predominantly attributable to an increase in the volume of interstitial cells (b) and results in an increase in the total arithmetic mean thickness of blood–gas barrier (c). In (d) the arithmetic mean thickness of the interstitium is shown. Statistical analyses are based on a two-way ANOVA taking the factors “bleomycin pre-treatment” and “mechanical ventilation” into account. Statistically significant differences after adjustment of the p level for multiple testing using Tukey correction is indicated as follows:*p < 0.05

Fig. 3

Representative ultrastructure of AE2 cells in healthy (H) and bleomycin (B)-pre-injured lungs subjected to different ventilation. In B/no_ventil, some AE2 cells were characterized by very large LB compared to H/no_ventil. The dashed line illustrates the region of the AE2 cell which is enlarged in Fig. 4. LB lamellar body, air alveolar airspace, nucl nucleus. Scale bar: 2 µm

Fig. 4

Electron-lucent multivesicular bodies, l-mvb, (arrows) were observed close to the apical plasma membrane. In B/PEEP1 some AE2 cells had a considerably high number of l-mvb compared to the other groups, e.g., H/no_ventil. B/ PEEP1 is a higher magnification from the AE2 cell illustrated in Fig. 3. Scale bar: 2 µm

Fig. 5

Stereological data characterizing AE2 cells and the intracellular surfactant pool. The number-weighted mean volume of the AE2 cells decreased with increasing PEEP during mechanical ventilation in healthy lungs but not in bleomycin-pre-injured lungs (a). In healthy lungs, mechanical ventilation was linked with a decrease in the volume of LB per AE2 cell a behavior which was not observed after bleomycin challenge (b). The number of LB per AE2 cell remained stable in healthy lungs but increased in bleomycin-pre-injured lungs after mechanical ventilation (c). In general, mechanical ventilation with increasing PEEP was associated with a decrease in the number-weighted mean volume of LB, a behavior which was much more pronounced in bleomycin-pre-injured lungs (d). Statistical analyses are based on a two-way ANOVA taking the factors “bleomycin pre-treatment” and “mechanical ventilation” into account. Statistically significant differences after adjustment of the p level for multiple testing using Tukey correction is indicated as follows:*p < 0.05

Fig. 6

Stereological data related to multivesicular bodies (mvb). a The volumes of electron-lucent multivesicular bodies (l-mvb) per AE2 cell are largest after bleomycin challenge and mechanical ventilation with PEEP = 1 cmH₂O. In (b) the volumes of electron-dense multivesicular bodies (d-mvb) are illustrated. Statistical analyses are based on a two-way ANOVA with the factors “bleomycin pre-treatment” and “mechanical ventilation”. Statistical significant differences after adjustment of the p level for multiple testing using Tukey correction is indicated as follows:*p < 0.05

Fig. 7

Expression of hydrophobic surfactant proteins B and C as well as lipid transporter Abca3. While bleomycin had no effect on SP-B RNA expression (a), both SP-C (b) and Abca3 (c) were downregulated. Mechanical ventilation did not cause further changes in gene expression. The quantification of RNA expression was based on the 2^△△CT- method and HPRT was used as the housekeeper gene. Western Blots and densitometry measurements were used to quantify protein expression of proSP-B (d) and the 16 kDa (e) as well as 21 kDa (f) variant of proSP-C in the homogenate of lavaged lungs. In (g) representative Western Blots of proSP-B are shown while h illustrates representative Western Blots of proSP-C. Statistical analyses are based on a two-way ANOVA with the factors “bleomycin pre-treatment” and “mechanical ventilation”. Statistical significant differences after adjustment of the p level for multiple testing using Tukey correction is indicated as follows:*p < 0.05; ns not significant

Fig. 8

Correlation between structural and lung mechanical data. The volume of electron-lucent multivesicular bodies (l-mvb) per AE2 cell demonstrates a positive correlation with the arithmetic mean thickness of the blood–gas barrier (a) and a negative correlation with the quasi-static compliance (b). The arithmetic mean thickness of the blood–gas barrier correlated negatively with the quasi-static compliance (c). Tissue elastance H, based on impedance data from forced oscillation perturbations at the conclusion of the mechanical ventilation demonstrated a positive correlation with the volume of electron-lucent multivesicular bodies (d). The tissue elastance data shown in d were taken from a previous publication (Albert et al. 2020). The Pearson correlation coefficient r and statistical significant p are given. Linear regression was used to fit a line to the measured data. The 95% confidence interval of the fitted line is given by the dashed borders