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. Jan-Mar 2019;9(1):2045894019826941.
doi: 10.1177/2045894019826941.

Analysis of Pulmonary Vascular Injury and Repair During Pseudomonas Aeruginosa Infection-Induced Pneumonia and Acute Respiratory Distress Syndrome

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Free PMC article

Analysis of Pulmonary Vascular Injury and Repair During Pseudomonas Aeruginosa Infection-Induced Pneumonia and Acute Respiratory Distress Syndrome

Ashley S Lindsey et al. Pulm Circ. .
Free PMC article

Abstract

Herein we describe lung vascular injury and repair using a rodent model of Pseudomonas aeruginosa pneumonia-induced acute respiratory distress syndrome (ARDS) during: 1) the exudative phase (48-hour survivors) and 2) the reparative/fibro-proliferative phase (1-week survivors). Pneumonia was induced by intratracheal instillation of P. aeruginosa strain PA103, and lung morphology and pulmonary vascular function were determined subsequently. Pulmonary vascular function was assessed in mechanically ventilated animals in vivo (air dead space, PaO2, and lung mechanics) and lung permeability was determined in isolated perfused lungs ex vivo (vascular filtration coefficient and extravascular lung water). At 48 hours post infection, histological analyses demonstrated capillary endothelial disruption, diffuse alveolar damage, perivascular cuffs, and neutrophil influx into lung parenchyma. Infected animals displayed clinical hallmarks of ARDS, including increased vascular permeability, increased dead space, impaired gas exchange, and decreased lung compliance. Overall, the animal infection model recapitulated the morphological and functional changes typically observed in lungs from patients during the exudative phase of ARDS. At 1 week post infection, there was lung histological and pulmonary vascular functional evidence of repair when compared with 48 hours post infection; however, some parameters were still impaired when compared with uninfected controls. Importantly, lungs displayed increased fibrosis and cellular hyperplasia reminiscent of lungs from patients during the fibro-proliferative phase of ARDS. Control, sham inoculated animals showed normal lung histology and function. These data represent the first comprehensive assessment of lung pathophysiology during the exudative and reparative/fibro-proliferative phases of P. aeruginosa pneumonia-induced ARDS, and position this pre-clinical model for use in interventional studies aimed at advancing clinical care.

Keywords: acute respiratory distress syndrome (ARDS); endothelial permeability; pulmonary edema; pulmonary endothelial cells.

Figures

Fig. 1.
Fig. 1.
Instillation of wild type P. aeruginosa strain PA103 (expressing T3SS effectors ExoU and ExoT) into the airways of wild type CD rats produces a dose–response effect on survival. PA103 was suspended into sterile normal saline solution and inoculated at the doses indicated in the figure. Animals were followed for 1 week post inoculation. These studies determined an infectious dose of 5 × 107 CFUs as the LD50, which was used in all subsequent experiments.
Fig. 2.
Fig. 2.
Microscopic evidence of the acute and fibro-proliferative phases of PA103-induced lung injury. Lungs were fixed and sectioned for H&E staining (panels (a) to (d), reference bar = 25 µm). (a) Lungs from control animals inoculated with saline solution alone were characterized by thin alveolar walls, clear alveolar spaces, and very few neutrophils, which were confined to the septal network. (b) Lungs from animals at 48 hours post PA103 inoculation were characterized by thickening of the alveolar walls, edema and hemorrhage, perivascular edema, accumulation of neutrophils within the alveolar airspaces, and vascular congestion. (c) Inset panel highlighting the presence of perivascular cuffing and cellular infiltrates. (d) Lungs from animals at 1 week post PA103 inoculation were characterized by alveolar wall thickening, with scant edema and hemorrhage, perivascular edema, or accumulation of neutrophils within the alveolar airspaces. (e) Quantitation of fluid accumulation in intra-acinar spaces. Airway instillation of PA103 increased the fraction of intra-acinar spaces that contained fluid as compared with the total intra-acinar spaces observed (VAF/VAT). **P = .001 when compared with all other groups by one-way ANOVA. (f) Quantification of vessels with perivascular cuffs. Airway instillation of PA103 increased the percentage of vessels with perivascular cuffs. *P = .027 compared with saline solution by one-way ANOVA. (g) Quantitation of fluid accumulation in perivascular spaces. Airway instillation of PA103 increased the fraction of perivascular cuff volume as compared with the total wall volume (vessel area plus cuff area, VC/VT). *P < .0001 compared with saline solution by one-way ANOVA. For panels (e) to (g), open circles represent measurements from individual lungs and closed circles indicate average ± SEM.
Fig. 3.
Fig. 3.
Evidence of extracellular matrix deposition and fibrotic lesions during PA103-induced lung injury. For panels (a) to (c), lungs were fixed and sectioned for trichrome staining, where blue staining represents regions of collagen deposition (reference bar = 20 µm). (a) Lungs from control animals inoculated with saline solution alone were characterized by thin alveolar walls and clear alveolar spaces. (b) Lungs from animals at 48 hours post PA103 inoculation were characterized by thickening of the alveolar walls, edema, increased cellular infiltrates, and evidence of extracellular matrix deposition and fibrotic lesions. (c) Lungs from animals at 1 week post PA103 inoculation were characterized by marked extracellular matrix deposition and dense fibrotic lesions indicative of injury and repair. (d) Assessment of hydroxyproline levels as a marker of collagen deposition. Lungs from animals at 1 week post PA103 inoculation displayed significantly higher levels of hydroxyproline compared with lungs from animals at 48 hours post infection. *P < .02 compared with acute by one-way ANOVA. Bars represent average ± SEM.
Fig. 4.
Fig. 4.
Ultrastructural imaging of distal lung parenchyma during PA103-induced lung injury. Lungs were fixed and sectioned for TEM (reference bar = 5 µm). (a) and (b) Lungs from control animals were characterized by a normal alveolar–capillary structure with thin alveolar walls and clear alveolar spaces. (c) and (d) Lungs from animals at 48 hours post PA103 inoculation were characterized by loss of inter-endothelial junctions with interstitial edema (star), pericapillary edema, and presence of red blood cells (asterisk) and inflammatory cells (arrow). Fibrinoid deposits reminiscent of hyaline membranes were observed in alveolar spaces (hashtag). Microvascular congestion within capillary lumen (Roleaux) and detachment of endothelium from the basal membrane (ellipse) were also evident. (e) and (f) Lungs from animals at 1 week post PA103 inoculation were characterized by basal membrane remodeling and deposition of extracellular matrix (chevron). There was little observable evidence of edema, red blood cells, or inflammatory cell infiltrates in the alveolar space. AS: airspace; CL: capillary lumen.
Fig. 5.
Fig. 5.
Functional evidence of pulmonary vascular permeability during PA103-induced lung injury. (a) Lungs from control animals (saline solution) or from PA103-inoculated animals (at 48 hours or 1 week post inoculation) were excised en bloc, and the filtration coefficient (Kf) was measured. Compared with controls, lungs from animals at 48 hours post PA103 inoculation displayed a significant increase in Kf, indicative of pulmonary vascular barrier compromise. Lungs from animals at 1 week post PA103 inoculation were not significantly different from control. **P < .0001 when compared with all other groups by one-way ANOVA. (b) Lungs from control animals (saline solution) or from PA103-inoculated animals (at 48 hours or 1 week post inoculation) were excised and EVLW was measured. Compared with controls, lungs from animals at 48 hours post PA103 inoculation displayed a significant increase in EVLW, indicative of pulmonary vascular barrier compromise. Lungs from animals at 1 week post PA103 inoculation were not significantly different from control animal lungs. **P = .009 when compared with all other groups by one-way ANOVA. Open circles represent measurements from individual lungs and closed circles indicate average ± SEM.
Fig. 6.
Fig. 6.
Functional evidence of lung gas exchange during PA103-induced lung injury. Control animals (saline solution), or PA103 inoculated animals (at 48 hours or 1 week post inoculation) were mechanically ventilated to allow measurement of lung gas exchange. (a) VD/VT measures areas of the lung that are ventilated but not perfused. Compared with controls, lungs from animals at 48 hours post PA103 inoculation displayed a significant increase in VD/VT, indicative of pulmonary vascular dysfunction. Lungs from animals at 1 week post PA103 inoculation displayed elevated VD/VT but were not significantly different from control animal lungs. **P < .0001 when compared with all other groups by one-way ANOVA. (b) PaO2 measures lung gas exchange capability. Compared with controls, lungs from animals at 48 hours and 1 week post PA103 inoculation displayed significant decreases in PaO2, indicative of impaired gas exchange. **P < .0001 when compared with all other groups by one-way ANOVA. Open circles represent measurements from individual lungs and closed circles indicate average ± SEM.
Fig. 7.
Fig. 7.
Functional assessment of lung mechanics during PA103-induced lung injury. Control animals (saline solution) and PA103-inoculated animals (at 48 hours or 1 week post inoculation) were mechanically ventilated to allow measurement of lung mechanics. (a) Dynamic compliance measures lung volume for a given applied pressure during periods of air flow. Compared with controls, lungs from animals at 48 hours and 1 week post PA103 inoculation displayed significant decreases in compliance, indicative of lung stiffening due to edema and fibrosis. *P < .0001 when compared with saline solution by one-way ANOVA. (b) Lung airway resistance measures the pressure required to open small airways (i.e., alveolar spaces). Compared with controls, lungs from animals at 48 hours post PA103 inoculation displayed a significant increase in small airway resistance, likely due to decreased dynamic compliance. Lungs from animals at 1 week post PA103 inoculation were not significantly different from control animal lungs. **P < .0001 when compared with all other groups by one-way ANOVA. (c) Airway resistance measures the pressure required to open large airways. Lungs from animals at 48 hours post PA103 inoculation were not significantly different from control animal lungs. Lungs from animals at 1 week post PA103 inoculation were significantly lower compared with control. *P = .006 when compared with saline solution by one-way ANOVA. Open circles represent measurements from individual lungs and closed circles indicate average ± SEM.
Fig. 8.
Fig. 8.
Functional assessment of mechanics in the distal lung parenchyma during PA103-induced lung injury. Control animals (saline solution) or PA103-inoculated animals (at 48 hours or 1 week post inoculation) were mechanically ventilated to allow measurement of mechanics in the distal lung parenchyma. (a) Tissue damping (G) measures the resistive forces of the lung parenchyma, which assesses air flow-independent distal airway elastic recoil. Compared with controls, lungs from animals at 48 hours and 1 week post PA103 inoculation displayed significantly increased tissue damping, indicative of lung stiffening due to edema and fibrosis. **P = .006 when compared with all other groups by one-way ANOVA. (b) Tissue elastance (H) measures the elastic forces of the lung parenchyma, indicating the amount of force needed to overcome the elastic recoil of the lung upon inspiration. Compared with controls, lungs from animals at 48 hours and 1 week post PA103 inoculation displayed significantly increased tissue elastance as a further indication of lung stiffening and fibrosis. **P = .006 when compared with all other groups by one-way ANOVA. Open circles represent measurements from individual lungs and closed circles indicate average ± SEM.

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