Endotoxin-Induced Emphysema Exacerbation: A Novel Model of Chronic Obstructive Pulmonary Disease Exacerbations Causing Cardiopulmonary Impairment and Diaphragm Dysfunction

Abstract

Chronic obstructive pulmonary disease (COPD) is a progressive disorder of the lung parenchyma which also involves extrapulmonary manifestations, such as cardiovascular impairment, diaphragm dysfunction, and frequent exacerbations. The development of animal models is important to elucidate the pathophysiology of COPD exacerbations and enable analysis of possible therapeutic approaches. We aimed to characterize a model of acute emphysema exacerbation and evaluate its consequences on the lung, heart, and diaphragm. Twenty-four Wistar rats were randomly assigned into one of two groups: control (C) or emphysema (ELA). In ELA group, animals received four intratracheal instillations of pancreatic porcine elastase (PPE) at 1-week intervals. The C group received saline under the same protocol. Five weeks after the last instillation, C and ELA animals received saline (SAL) or E. coli lipopolysaccharide (LPS) (200 μg in 200 μl) intratracheally. Twenty-four hours after saline or endotoxin administration, arterial blood gases, lung inflammation and morphometry, collagen fiber content, and lung mechanics were analyzed. Echocardiography, diaphragm ultrasonography (US), and computed tomography (CT) of the chest were done. ELA-LPS animals, compared to ELA-SAL, exhibited decreased arterial oxygenation; increases in alveolar collapse (p < 0.0001), relative neutrophil counts (p = 0.007), levels of cytokine-induced neutrophil chemoattractant-1, interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and vascular endothelial growth factor in lung tissue, collagen fiber deposition in alveolar septa, airways, and pulmonary vessel walls, and dynamic lung elastance (p < 0.0001); reduced pulmonary acceleration time/ejection time ratio, (an indirect index of pulmonary arterial hypertension); decreased diaphragm thickening fraction and excursion; and areas of emphysema associated with heterogeneous alveolar opacities on chest CT. In conclusion, we developed a model of endotoxin-induced emphysema exacerbation that affected not only the lungs but also the heart and diaphragm, thus resembling several features of human disease. This model of emphysema should allow preclinical testing of novel therapies with potential for translation into clinical practice.

Keywords: collagen fiber; diaphragm dysfunction; emphysema; lung mechanics; pulmonary arterial hypertension.

Figure 1

(A) Schematic flow chart and (B) timeline of the study design. Animals were randomly assigned into two main groups: control (C) and emphysema (ELA). In the ELA group, animals received four intratracheal instillations of pancreatic porcine elastase (PPE) at 1-week intervals. The C group received saline alone using the same protocol. Five weeks after the last instillation, C and ELA animals received saline (SAL) or E. coli LPS (LPS) intratracheally.

Figure 2

(A) Mean linear intercept (Lm). (B) Central moments of mean linear intercept (D2 of Lm). (C) Heterogeneity index (β) and (D) representative photomicrographs of lung parenchyma stained with hematoxylin-eosin (HE) (400× magnification). Note alveolar space enlargement in the ELA groups (horizontal arrows) and increased alveolar collapse after exacerbation (sloping arrows) (ELA-LPS group). C, control; ELA, emphysema; SAL, animals treated with saline; LPS, animals treated with E. coli LPS. Values are means + SD of six animals in each group.

Figure 3

Transmission electron microscopy of lung parenchyma in control (C) and animals exposed to saline (A–C) or E. coli LPS (D–F), as well as in emphysema (ELA) rats exposed to saline (G–I) or E. coli LPS (J–L). Note preserved alveolar walls (AW), integrity of endothelial cells (EC) of the alveolar–capillary membrane (ACM), and lamellar bodies (LB) of alveolar epithelial cells (AEC) in the C-SAL group. C-LPS animals show collapse of AW with increased neutrophil (N) and macrophage (M) counts, endothelial-cell damage (ED), detachment of AEC with increased permeability of the alveolar–capillary membrane, and interstitial edema (IE). ELA-SAL animals show alveolar wall disruption (AWD) and irregularity (AWI), inflammation, and destruction of elastic fibers (EF). The pattern of inflammation involves increased numbers of neutrophils (N) and macrophages (M). These cells produce proteases involved in tissue destruction and release local and systemic inflammatory mediators, which amplify inflammation and structural disruption. Note detachment of AEC and reduction in LB. After emphysema exacerbation, there is an increase in neutrophil (N) and macrophage (M) response, increased detachment of AEC, loss of LB, and ED, causing increased permeability of the alveolar–capillary membrane and IE due to surfactant abnormalities.

Figure 4

Levels of (A) keratinocyte-derived chemokine (cytokine-induced neutrophil chemoattractant [CINC]-1, a rat analog of interleukin-8), (B) interleukin (IL)-1β, (C) IL-6, (D) tumor necrosis factor (TNF)-α, and (E) vascular endothelial growth factor (VEGF) in lung tissue. C, control; ELA, animals treated with intratracheal instillations of elastase; SAL, saline; LPS, E. coli lipopolysaccharide. Values are median (interquartile range) of six animals in each group.

Figure 5

Collagen fiber content and representative photomicrographs of (A) alveolar septa, (B) airways, and (C) pulmonary vessel wall stained with the Picrosirius-polarization method (collagen fibers). C, control; ELA, animals treated with intratracheal instillations of elastase; SAL, saline; LPS, E. coli lipopolysaccharide. Values are mean ± SD of six animals in each group.

Figure 6

(A) Elastic fiber content in alveolar septa and (B) representative photomicrographs of the lung parenchyma stained with Weigert’s resorcin fuchsin method with oxidation (elastic fibers). Red arrows: elastic fibers are stained in black. C, control; ELA, animals treated with intratracheal instillations of elastase; SAL, saline; LPS, E. coli lipopolysaccharide. Values are mean ± SD of six animals in each group.

Figure 7

Echocardiography. (A) right ventricular end-diastolic area, (B) diastolic right ventricular wall thickness, (C) ejection fraction, (D) heart rate, (E) PAT/PET ratio, and (F) representative images of pulmonary blood flow. C, control; ELA, animals treated with intratracheal instillations of elastase; SAL, saline; LPS, E. coli lipopolysaccharide. Values are mean ± SD of six animals in each group.

Figure 8

Diaphragm ultrasound. (A) diaphragm thickening fraction, (B) diaphragm excursion, and (C) ultrasonographic view of diaphragmatic excursion (DE) and its thickening fraction (TF), calculated by end inspiratory thickness (EIT) – end-expiratory thickness (EET)/end-expiratory thickness × 100. C, control; ELA, animals treated with intratracheal instillations of elastase; SAL, saline; LPS, E. coli lipopolysaccharide. Values are mean ± SD of six animals in each group.

Figure 9

Transmission electron microscopy of diaphragm in control (C) animals treated with saline (A–C) or E. coli LPS (D–F), as well as in emphysema (ELA) animals treated with saline (G–I) or E. coli LPS (J–L). Note I-band integrity and preserved myosin and mitochondria in C-SAL group. The C-LPS group shows discrete changes in these structures. The ELA-SAL group exhibits disorganized I-bands, glycogen accumulation in sarcoplasm, loss of myosin content in myofibrils, subsarcolemmal mitochondrial aggregates, and thickened Z lines. The ELA-LPS group shows even more intense changes in these structures.