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. 2018 Jun;58(6):696-705.
doi: 10.1165/rcmb.2017-0168OC.

Bronchiolitis Obliterans and Pulmonary Fibrosis After Sulfur Mustard Inhalation in Rats

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Free PMC article

Bronchiolitis Obliterans and Pulmonary Fibrosis After Sulfur Mustard Inhalation in Rats

Matthew D McGraw et al. Am J Respir Cell Mol Biol. .
Free PMC article

Abstract

Inhalation of powerful chemical agents, such as sulfur mustard (SM), can have debilitating pulmonary consequences, such as bronchiolitis obliterans (BO) and parenchymal fibrosis (PF). The underlying pathogenesis of disorders after SM inhalation is not clearly understood, resulting in a paucity of effective therapies. In this study, we evaluated the role of profibrotic pathways involving transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) in the development of BO and PF after SM inhalation injury using a rat model. Adult Sprague-Dawley rats were intubated and exposed to SM (1.0 mg/kg), then monitored daily for respiratory distress, oxygen saturation changes, and weight loss. Rats were killed at 7, 14, 21, or 28 days, and markers of injury were determined by histopathology; pulmonary function testing; and assessment of TGF-β, PDGF, and PAI-1 concentrations. Respiratory distress developed over time after SM inhalation, with progressive hypoxemia, respiratory distress, and weight loss. Histopathology confirmed the presence of both BO and PF, and both gradually worsened with time. Pulmonary function testing demonstrated a time-dependent increase in lung resistance, as well as a decrease in lung compliance. Concentrations of TGF-β, PDGF, and PAI-1 were elevated at 28 days in lung, BAL fluid, and/or plasma. Time-dependent development of BO and PF occurs in lungs of rats exposed to SM inhalation, and the elevated concentrations of TGF-β, PDGF, and PAI-1 suggest involvement of these profibrotic pathways in the aberrant remodeling after injury.

Keywords: PAI-1; bronchiolitis obliterans; lung fibrosis; sulfur mustard; transforming growth factor-β.

Figures

Figure 1.
Figure 1.
Kaplan-Meier survival curves after sulfur mustard (SM) exposure. (A) Various exposure doses of SM inhalation in rats at sea level have a dose-dependent lethality over 28 days. Black line = 0.5 mg/kg; purple line = 0.75 mg/kg; blue line = 1.0 mg/kg; green line = 1.4 mg/kg; red line = 3.8 mg/kg. (B) Kaplan-Meier survival curve at Denver altitude (5,280 ft) (blue line) and at the Aberdeen Proving Ground (sea level) (black line) with 1.0 mg/kg SM exposure. Lethality was not significantly different between altitude and sea level (P = 0.98). In both models, mortality occurred in a biphasic mode, with the initial onset of death occurring acutely within the first 7 days after exposure, followed by a secondary progressive decline beginning 14 days after exposure.
Figure 2.
Figure 2.
Lung function testing in rats exposed to SM (1.0 mg/kg) compared with naive control rats at 7, 14, 21, and 28 days. (A) Mean total lung resistance (R; cm H2O · s/ml) increased over time after SM exposure. At Days 7 and 14, R was not different from that in naive control rats, but by Day 21, R was significantly elevated (P < 0.0001), and it remained elevated at Day 28 (P < 0.0001) compared with naive levels. (B) Mean Newtonian resistance (Rn; cm H2O · s/ml) increased over time after SM exposure. At Days 7 and 14, Rn was not different from that in naive control rats, but by Day 21, Rn was significantly elevated (P < 0.05), and it remained elevated at Day 28 (P < 0.05) compared with naive levels. (C) Mean tissue damping (G; cm H2O/ml) increased over time after SM exposure. At Days 7 and 14, G was not different from that of naive control rats, but by Day 21, G was significantly elevated (P < 0.0001), and it remained elevated at Day 28 (P < 0.0001) compared with naive levels. (D) Mean total lung compliance (C; ml/cm H2O) decreased over time after SM exposure. By Day 14, C was significantly decreased (P < 0.0001) compared with that of naive control rats, and it continued to decrease further at Day 21 (P < 0.0001) and Day 28 (P < 0.0001). Data are presented as mean ± SEM. P values were derived using ANOVA with Tukey’s post hoc test. **P < 0.01, ****P < 0.0001.
Figure 3.
Figure 3.
Histopathology of distal rat lung bronchioles after SM inhalation (1.0 mg/kg) exposure compared with that of naive control rats. (A) Naive rat bronchiole showing normal lung histology (Masson’s trichrome stain; magnification, ×10). (B) Representative rat bronchiole 28 days after SM inhalation, with significant collagen deposition (blue) and luminal narrowing, consistent with constrictive type bronchiolitis obliterans (Masson’s trichrome stain; magnification, ×10). (C) Higher-magnification view of naive control rat bronchiole showing normal histopathological pattern (Masson’s trichrome stain; magnification, ×20). (D) Higher-magnification view of a representative distal rat bronchiole 28 days after SM with extensive collagen deposition (blue), hypercellularity, and greater than 90% luminal narrowing, consistent with constrictive type bronchiolitis obliterans (Masson’s trichrome stain; magnification, ×20). Dot-dashed circle estimates the normal airway luminal circumference (red dashed line) (Masson’s trichrome stain). Scale bars: 200 μm (A and B) and 100 μm (C and D).
Figure 4.
Figure 4.
Histopathology of lung parenchyma after SM inhalation (1.0 mg/kg) exposure over time (14–28 d) compared with naive control animals. (A) Naive rat lung showing normal parenchymal structure. (B) Representative rat lung parenchymal section 14 days after SM exposure with moderate amounts of collagen deposition (blue), showing nearly 30% of parenchyma with collagen staining (inset). (C) Representative rat lung parenchymal section 21 days after SM exposure with progressive increase of interstitial infiltrates and collagen deposition, with nearly 50% of alveolar cross-sectional area occluded with fibrotic material (inset). (D) Representative rat lung parenchymal section 28 days after SM exposure showing diffuse parenchymal fibrosis. Greater than 50% collagen deposition (blue) is present within the subpleural regions (inset). All images show Masson’s trichrome stain; magnification of main images, ×4; magnification of insets, ×40. Scale bars: 500 μm.
Figure 5.
Figure 5.
Modified Ashcroft scoring for parenchymal fibrosis in rat lungs after SM (1.0 mg/kg) exposure compared with naive control animals. (A) All individual lung lobes were affected 17–28 days (pooled data) after SM inhalation exposure, with the highest Ashcroft scores, and thus the most severe fibrosis, in the RUL and RLL (****P < 0.0001 compared with naive that had 0 scores). (B) RLL Ashcroft scores increased over time after SM exposure, with worse scores at 24–28 days over earlier time points. Data are presented as mean ± SEM; ANOVA with Tukey’s post hoc test. LLL = left lower lobe; RA = right accessory; RLL = right lower lobe; RML = right middle lobe; RUL = right upper lobe.
Figure 6.
Figure 6.
Transforming growth factor-β1 (TGF-β1), platelet-derived growth factor A/B (PDGF-A/B), and PAI-1 in lung, BAL fluid (BALF) and plasma after SM inhalation exposure. (A) TGF-β1 concentrations in lung homogenates were significantly elevated at 21 days (P < 0.01) and 28 days (P < 0.05) after SM exposure compared with naive control concentrations. (B) TGF-β1 concentrations in BALF were nonsignificantly elevated at 21 and 28 days after SM exposure compared with naive control concentrations. (C) PDGF-A/B concentrations in total lung homogenates were significantly elevated at 21 days (P < 0.0001) compared with naive control concentrations. (D) PDGF-A/B concentrations in BALF were nonsignificantly elevated at 21 and 28 days after SM exposure compared with naive control concentrations. (E) PAI-1 concentrations in plasma were significantly elevated at 28 days (P < 0.05) after SM compared with naive control concentrations. (F) PAI-1 concentrations in BALF were significantly elevated at 28 days (P < 0.001) compared with naive control concentrations. Data are presented as mean ± SEM; ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 7.
Figure 7.
Immunofluorescence staining of lung sections for TGF-β1 and platelet-derived growth factor receptor-α (PDGFR-α) after SM inhalation exposure (1.0 mg/kg) compared with that in naive control specimens. (A) Naive rat lung showing a minimal amount of TGF-β1 (green) staining within the lung parenchyma. (B) Representative rat lung parenchymal section 21 days after SM exposure showing increased TGF-β1 (green) staining in alveolar regions that correspond to fibrotic areas within the lung. (C) Naive rat lung showing a minimal amount of PDGFR-α (red) staining within the lung parenchyma. (D) Representative rat lung parenchymal section 21 days after SM exposure showing increased PDGFR-α (red) staining in alveolar regions that correspond to fibrotic areas within the lung. Magnification of main images, ×4; magnification of insets, ×20. Scale bars: 500 μm.

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