Interruption of intermittent hypoxia attenuates the severity of pulmonary fibrosis in male mice

Abstract

Obstructive sleep apnoea (OSA) leading to chronic intermittent hypoxia (CIH) is a common comorbidity associated with idiopathic pulmonary fibrosis (IPF), a progressive fatal disease characterized by adverse lung remodeling and parenchymal stiffening. In the mouse model of lung fibrosis induced by intra-tracheal instillation (ITI) of bleomycin, we have previously shown that CIH exacerbates fibrosis, similar to pre-exposure to ITI. It has been suggested that correction of OSA and CIH with positive airway pressure may have a beneficial effect on the progression of fibrosis in IPF patients. Therefore, we designed this study to determine the benefits of stopping CIH in mice pre-exposed to CIH for 3 weeks prior to bleomycin ITI. Interruption of CIH exposure was associated with less diffuse lung fibrosis, reduced ratio of damaged area, alveolar destruction, collagen deposition, and fibrosis score. Interruption of CIH exposure significantly reduced macrophage density in fibrotic areas without significant changes in either lymphocyte or neutrophil density. In conclusion, this study demonstrates that early treatment of OSA-associated CIH can limit or reduce the development of severe forms of pulmonary fibrosis associated with a reduction in macrophage infiltrate and supports the beneficial effect of CIH exposure interruption on ongoing fibrosis severity.

Keywords: intermittent hypoxia; lung damages; macrophages; obstructive sleep apnoea; pulmonary fibrosis.

FIGURE 1

Experimental design and body mass evolution. (a) Mice were exposed to chronic intermittent hypoxia (CIH; 30 cycles/hour, 8 h/day, nadir 7% O₂) for 21 days. Subsequently, they received an intratracheal instillation (IT) of 3.5 UI/g of bleomycin (BLM) and then half of the animals were left in CIH for another 21 days (CIH/BLM‐CIH; n = 5) whereas the other half were left in air (CIH/BLM‐Air; n = 5). (b) Body mass was monitored for all mice and was represented by percentage of mass loss from the day of IT instillation of BLM (median ± IR).

FIGURE 2

Pulmonary architecture. (a) Cartography of mouse lungs stained with Hematoxylin Eosin. (b) Illustration of injured area (×40 magnification). (c) Histologic illustration of collagen deposition (Masson's Trichrome staining, x40 magnification). (a, b, and c) A representative image of mice lung from the CIH/BLM‐CIH (n = 5) and CIH/BLM‐Air (n = 5) groups was shown. (d) Quantification of lung injury area reported to the total lung surface, (e) Mean Linear Intercept (MLI) measured on five microscopic images obtained at ×40 magnification of Hematoxylin Eosin‐stained lung biopsy sections, (f) Percentage of collagen occupation on lungs stained with Masson's trichrome of all the mice from the CIH/BLM‐CIH (n = 5) and CIH/BLM‐Air (n = 5) groups. (g) Histogram bars represent the mean of Aschroft scores with the intra proportion of different fibrotic stages from 1 (normal lung, no fibrotic burden and small fibers) to 5 (completely remodeled lung, large continuous fibrotic masses >50%). Scoring was performed on five images each from CIH/BLM‐CIH (n = 5) and CIH/BLM‐Air (n = 5) mice.

FIGURE 3

Pulmonary inflammatory response. (a, b, and c) Analysis of differential inflammatory cells by immunohistochemistry for total F4‐80 (a), Ly6G (b), and CD4 positive cells (c) performed on 5 μm lung biopsy sections. A representative image of mice from the CIH/BLM‐CIH (n = 5) and CIH/BLM‐Air (n = 5) groups is shown. (d) Semi‐quantification of F4‐80, Ly6G, and CD4 positive cells counted in lung sections from CIH/BLM‐CIH (n = 5) and CIH/BLM‐Air (n = 5) mice. (e) Proportions of macrophages (F4‐80), lymphocytes (CD4), and neutrophils (Ly6G) in the CIH/BLM‐CIH (n = 5) and CIH/BLM‐Air (n = 5) groups.