Bleomycin induced lung fibrosis increases work of breathing in the mouse

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Abstract

Bleomycin induces a transient lung fibrosis in mice that has been used to investigate mechanisms related to idiopathic pulmonary fibrosis. Our aim was to determine a sensitive method for assessing lung function in bleomycin treated mice that correlated with the degree of lung fibrosis as measured by collagen immunohistochemistry. Bleomycin (2 U/kg) or saline was intratracheally microsprayed to male C57BL/6 mice under isoflurane anesthesia. Lung function (single compartment model, constant phase model, and work of breathing) was assessed using the flexiVent system, and after euthanasia lungs were inflated with formalin in situ for histological analysis. The lung fibrosis histopathology score for the bleomycin treated animals on day 21 was indicative of mild-to-moderate fibrosis (Saline treated control: 0 ± 0, Bleomycin treated: 4.9 ± 0.4). There were at least three large areas of fibrosis in the peribronchial alveolar regions of the lung, but less than 50% of each lung was affected by fibrosis. Although changes in lung function were less obvious, volume normalized dynamic work of breathing measured at 30 ml/kg tidal volume (Saline treated control: 9.2 ± 0.1 J/l, Bleomycin treated: 10.6 ± 0.3 J/l) and the oscillatory mechanics constant phase model parameter tissue elastance (H; Saline treated control: 31 ± 2 cm H2O/ml, Bleomycin treated: 38 ± 3 cm H2O/ml) were significantly increased on day 21. The work of breathing (r = 0.83) correlated slightly better with fibrosis histopathology score than H (r = 0.64). Work of breathing can detect decrements in lung function due to pulmonary fibrosis, correlates well with the amount of collagen in the lungs, and may be a more sensitive quantitative measure of efficacy for drugs being developed to treat pulmonary fibrosis.

Introduction

In fibrotic lungs, the pressure-volume (PV) curve and hence the mechanical properties of the lung change in a manner that implies alteration in collagen elasticity. Collagen is an important load-bearing element within the lung. Total collagen content in the fibrotic lung increases and is accompanied by a decrease in lung function [1]. The relationship between the molecular organization of collagen fibers and the in vivo PV curve is not fully understood. In the bleomycin induced mouse model of pulmonary fibrosis, the small size of the mouse and the variable distribution of fibrotic foci in the lungs present significant challenges for the assessment of lung function. Histological analysis is a robust qualitative method for assessing lung fibrosis in this model; however, measuring lung function provides a quantitative assessment that may have an advantage in detecting improvement in lung stiffness (fibrosis) before it may become overtly evident histologically. Our aim was to determine a sensitive method for assessing lung function in bleomycin treated mice that correlated with the degree of lung fibrosis as measured by collagen immunohistochemistry.

Lung function data were calculated in three ways (the single compartment model, constant phase model, and work of breathing) using a flexiVent computer controlled ventilator. Dynamic PV curves measured by inflating and deflating the lungs (lung PV loops) can be used to calculate the work performed in ventilating the lung, since the product of pressure and volume is a unit of work. This study demonstrated that of the lung function parameters measured in the bleomycin induced mouse model of pulmonary fibrosis, the volume normalized work of breathing measured at 30 ml/kg was the most significantly increased and correlate most strongly with the lung fibrosis histopathology score.

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Animals

Male C57BL/6 mice weighing 22–24 g on day 0 (7–9 weeks old, Jackson Laboratories, n = 7–8 animals per group) were used in this study. Animal care and experimental procedures used in this study were approved by the Roche Animal Care and Use Committee (RACUC, Nutley, NJ, USA) which is a facility accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC).

Bleomycin induced mouse model of pulmonary fibrosis

Bleomycin sulphate (MP Biomedicals, Solon, OH, USA) was dissolved in 0.9% saline at a concentration of

Effects on body weight

A significant decrease in body weight after 2 U/kg, i.t. bleomycin administration was initially observed (Fig. 1). The decrease in body weight in the bleomycin treated group stabilized after day 7 and daily increases in body weight resumed in the bleomycin treated group after day 9. The average body weight of the bleomycin treated group (25.1 ± 0.6 g) remained significantly lower that the saline control group (26.8 ± 0.4 g) on day 21. None of the 2 U/kg bleomycin treated animals died before day

Discussion

This study demonstrated that among the lung function parameters we have measured in the bleomycin induced mouse model of pulmonary fibrosis, the volume normalized work of breathing measured at 30 ml/kg was the most significantly increased and best correlate of the lung fibrosis histopathology score. Histological analysis is a robust but qualitative method to assess lung fibrosis. While lung function is not as robust of an indicator of pulmonary fibrosis, improvements in lung function can be

Conflicts of interest

All authors are full-time employees of the Hoffmann – La Roche Inc.

Acknowledgements

The authors would like to thank Dr. Martin Kolb (McMaster University) for his stimulating discussion on the applicability of this work. We would also like to thank Lorena Renteria, Jacob LaStant, and John Woods for their assistance with the preparation and harvest of the lung tissues.

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