Full Length ArticleSystemic bone loss, impaired osteogenic activity and type I muscle fiber atrophy in mice with elastase-induced pulmonary emphysema: Establishment of a COPD-related osteoporosis mouse model
Introduction
Chronic obstructive pulmonary disease (COPD) demonstrates skeletal muscle and bone disorders as extrapulmonary lesions and is a risk factor for osteoporosis and sarcopenia [[1], [2], [3], [4], [5], [6], [7], [8]]. Patients diagnosed with COPD have a high prevalence of osteoporosis [9]. The prevalence of vertebral fractures in COPD patients is 24 to 63% [10], and thoracic vertebral fractures occur more often than lumbar vertebral fractures [11]. In addition, COPD is the most frequent disease as a cause of secondary osteoporosis in men [12].
Chronic inflammation due to many factors occurs in the lungs of patients with COPD, and irreversible loss of alveolar surface and depletion of lung elastin, which are pulmonary emphysema findings, have been identified [13]. There is also a clinical phenotype called combined pulmonary fibrosis and emphysema (CPFE), which is characterized by the coexistence of pulmonary fibrosis and emphysema [14], but emphysema volume has especially been shown to be strongly related to vertebral bone mineral density (BMD) [15]. Almost all of the pathological mechanisms of COPD/emphysema-related osteoporosis remain unknown, although it is suggested that COPD/emphysema is related to bone.
The problem in investigating the pathological mechanism of COPD-related osteoporosis is that various factors exist that affect bone metabolism, such as smoking [16], body weight [17], COPD exacerbation [18], use of steroids [19], and sarcopenia [20]. In particular, sarcopenia and body weight loss in COPD subjects are related to excessive energy expenditure by respiratory muscles, systemic inflammation, nutrition disability and physical inactivity. Analysis excluding these factors is difficult in humans, and analysis in an animal model is desired; however, there is currently an absence of an appropriate animal model for COPD/emphysema-related osteoporosis patients.
Several pulmonary emphysema models exist [21], such as smoking exposure models, transgenic animals, and elastase-induced emphysema models. The smoking exposure model is the one most commonly used as a COPD animal model, and it demonstrates the deterioration of bone quality [22]. However, there is no unified experimental protocol [23]. In addition, the degree of emphysema is mild; therefore, it may be the influence of smoking exposure rather than emphysema causing bone to degrade. Problematically, not only weight but also fat and muscle mass decrease systemically over time [24]. In transgenic animals, we must consider the effect of genetic modifications on bone. An elastase-induced emphysema model is used to initiate the inflammatory response seen in COPD and to induce an inflammatory response to make it permanent [23]. The greatest advantage is that we can control the severity of pulmonary emphysema depending on the elastase dosage. Therefore, we considered it appropriate to use this model to exclude factors influencing bone metabolism (smoking, body weight loss, muscle mass loss) and to investigate the influence of COPD/emphysema on bone metabolism.
It is necessary for us develop a mouse model to elucidate the pathological mechanisms of a COPD/emphysema-related osteoporosis. We planned the following study, utilizing the elastase-induced emphysema model to establish an appropriate animal model for patients with COPD/emphysema-related osteoporosis. The purposes of our study are 1) to verify the elastase dosage that induces the emphysema without complications (weakness and body weight loss) and 2) to evaluate the bones and muscles of the pulmonary emphysema model mice. In addition, this study establishes a mouse model of patients with COPD/emphysema-related osteoporosis.
Section snippets
Mice
The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of the University of Occupational and Environmental Health (approval number, AE11-014). A total of 56 eight-week-old male C57BL/6 J mice (approximately 20 g) were purchased from Charles River Japan (Tokyo, Japan) and acclimated for 4 weeks under standard laboratory conditions (temperature 24 ± 1 °C, humidity 55 ± 5%). All of the mice were housed in similarly designed cages. The light/dark cycle was
Body weight
The experimental protocol design is shown in Fig. 1A. Seven of the 14 mice in the PPE 0.25 group were euthanized after a few days due to weakness. Four of the remaining 7 mice showed body weight losses of 15% or more at day 7 after intratracheal administration; thus, the experiment was stopped for the PPE 0.25 group. There were no significant differences in body weight at each week among the Control, PPE 0.025, and PPE 0.1 groups (Fig. 1B).
Lung histomorphometry
The effect of PPE administration into the trachea on
Discussion
This study was performed to establish a COPD/emphysema-related osteoporosis mouse model for elucidating the pathological mechanism of COPD/emphysema-related osteoporosis, and the following results were observed according to the purposes of this study. 1) PPE 0.025 U and PPE 0.1 U were the appropriate doses (Fig. 1 B–D) when we examined the elastase dose that induced emphysema without complications. Therefore, the bone and muscle data were compared among the PPE 0.1, PPE 0.025 and Control
Conclusions
The bone and muscle phenotypes in the mice with elastase-induced emphysema were highly similar to those previously reported in patients with COPD. We believe that the mice described in this experimental protocol will be accepted as a COPD/emphysema-related osteoporosis model.
The following is the supplementary data related to this article.
Disclosures
None of the authors have conflicts of interest to disclose.
Acknowledgments
The authors greatly appreciate the technical assistance of Nao Terada (Department of Orthopaedic Surgery), Minako Nimura (Department of Orthopaedic Surgery) and Ryoko Maekado (Shared-Use Research Center) from the University of Occupational and Environmental Health. This work was supported, in part, by a Grant-in-Aid for Scientific Research (JSPS KAKENHI Grant Number JP 16H07390) and Japan Osteoporosis Society 2018 Research Encouragement Award.
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Present/permanent address: Department of Orthopaedic Surgery, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, 807-8555, Japan.