Chronic intermittent hypoxia worsens bleomycin-induced lung fibrosis in rats

Abstract

Obstructive sleep apnea (OSA) has been linked to increased mortality in pulmonary fibrosis. Its key feature, chronic intermittent hypoxia (CIH), can lead to oxidative stress and inflammation, known to lead to fibrotic pathology in other organs. We tested the effects of CIH in an animal model of bleomycin-induced lung fibrosis. Sprague Dawley rats were instilled intratracheally with bleomycin (Blm) or saline (Sal), and exposed to CIH or normal air (Norm) for 9 or 30 days. Pulmonary function was tested and lungs were harvested for histological and molecular analyses. In Blm-treated animals, 30days of CIH compared to Norm increased total lung collagen content (p=0.008) and reduced Quasi-static lung compliance (p=0.04). CIH upregulated lipid peroxidation and increased NF-κB activation, IL-17 mRNA and Col1α1 mRNA expression. Our results indicate that following Blm-induced lung injury, CIH amplifies collagen deposition via oxidative and inflammatory pathways, culminating in stiffer lungs. Thus, OSA may augment fibrosis in patients with interstitial lung disease.

Keywords: Fibrosis; Hypoxia; Intermittent; Lung/immunology/metabolism/pathology; Obstructive; Sleep apnea.

Figure 1. Experimental Protocol

Animals were given a single intratracheal instillation of Blm or saline on Day 0. CIH or Normoxia exposures commenced on Day 5 after Blm. Endpoints were collected on Day 14 or Day 35 after Blm instillation. i.t. = intratracheal instillation of Blm.

Figure 2. 30 days of CIH and Blm increased total right lung collagen content

Total hydroxyproline content (μg) in lung tissue at day 14 (A) and day 35 following Blm (B).

Figure 3. Blm and 30 days of CIH increased fibrotic lung pathology

(A–H): Representative Masson Trichrome stain images (20×) of lung parenchyma in Sal/Norm (A, B), Sal/CIH (C, D), Blm/Norm (E, F) and Blm/CIH – treated (G, H) animals on Day 14 (A, C, E, G) and Day 35 (B, D, F, H) following Blm instillation; black arrows pointing towards areas of alveolar wall thickening (blue colour) in Sal/CIH – treated animals at Day 14 (C) and Day 35 (D), as well as dense fibrotic areas in Blm/CIH, 35 days after Blm injury (H). (J, K) Ashcroft scores of Masson Trichrome stained sections on Day 14 (J) and Day 35 following Blm instillation (K). Scale bar (Panel A): 100 μm.

Figure 4. Thirty days of CIH following Blm injury increased average area of α-Smooth Muscle Actin positive staining relative to total lung parenchymal tissue area

(A–D): Representative α-SMA immunostained images of Sal/Norm (A), Sal/CIH (B), Blm/Norm (C) and Blm/CIH (D) animals, 35 days after Sal or Blm treatment (20×). Emphysema-like changes are present in CIH treated animals (B, D) and increased foci of α-SMA+ staining (D; arrows) are seen in the lungs of Blm/CIH treated rats. Scale bar (shown in Panel A): 100 μm. (E): Average α-SMA+ region area (μm²). (F): Average SMA+ region area divided by total parenchymal tissue area in the microscope field, to account for presence of “emphysema-like” areas. Each symbol represents the average of all images (≥15) from one rat.

Figure 5. CIH leads to pulmonary function deficits following Blm injury

(A, B): Representative Pressure-Volume loops from Cst measurement in the indicated treatment groups; line segments indicate slope of the inspiratory curve at the point of half-maximum inflation (circles). (D–F): Quasi-static compliance (Cst) (C, D) and Parenchymal Tissue Elastance (H) (E, F) were measured on the indicated days after Blm instillation.

Figure 6. CIH did not affect the Blm-induced decline in lung volumes

Inspiratory Capacity (A, B) and Forced Vital Capacity (C, D) as a fraction of total lung capacity (TLC), and TLC (E, F) normalized to rat body weight were measured at the indicated days after bleomycin.

Figure 7. Prolonged CIH enhances Blm – induced oxidative stress and NF-κB activation in lung tissue

Total malondialdehyde (MDA) content (A, B) and NF-κB activity (C, D) were measured in lung tissue on day 14 (A, C) or day 35 (B, D) after Blm instillation. 30 days of CIH exposure enhanced Blm – induced increase in MDA content and led to increased NF-κB activity.

Figure 8. Prolonged CIH iIncreased IL-17 and CCN-2 mRNA expression

IL-17 (A, B) and CCN-2 (C, D) mRNA expression in lung tissue were measured by qPCR on day 14 (A, C) or day 35 (B, D) after Blm instillation. A.U: Arbitrary Units.

Figure 9. CIH increased Col1α1 and Col5α1 mRNA expression

Col1α1 (A, B) and Col5α1 (C, D) mRNA expression in lung tissue was measured by qPCR on day 14 (A, C) or day 35 (B, D) after Blm instillation. A.U: Arbitrary Units.