Antenatal Endotoxin Induces Dysanapsis in Experimental Bronchopulmonary Dysplasia

Abstract

Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, is characterized by impaired lung development with sustained functional abnormalities due to alterations of airways and the distal lung. Although clinical studies have shown striking associations between antenatal stress and BPD, little is known about the underlying pathogenetic mechanisms. Whether dysanapsis, the concept of discordant growth of the airways and parenchyma, contributes to late respiratory disease as a result of antenatal stress is unknown. We hypothesized that antenatal endotoxin (ETX) impairs juvenile lung function as a result of altered central airway and distal lung structure, suggesting the presence of dysanapsis in this preclinical BPD model. Fetal rats were exposed to intraamniotic ETX (10 μg) or saline solution (control) 2 days before term. We performed extensive structural and functional evaluation of the proximal airways and distal lung in 2-week-old rats. Distal lung structure was quantified by stereology. Conducting airway diameters were measured using micro-computed tomography. Lung function was assessed during invasive ventilation to quantify baseline mechanics, response to methacholine challenge, and spirometry. ETX-exposed pups exhibited distal lung simplification, decreased alveolar surface area, and decreased parenchyma-airway attachments. ETX-exposed pups exhibited decreased tracheal and second- and third-generation airway diameters. ETX increased respiratory system resistance and decreased lung compliance at baseline. Only Newtonian resistance, specific to large airways, exhibited increased methacholine reactivity in ETX-exposed pups compared with controls. ETX-exposed pups had a decreased ratio of FEV in 0.1 second to FVC and a normal FEV in 0.1 second, paralleling the clinical definition of dysanapsis. Antenatal ETX causes abnormalities of the central airways and distal lung growth, suggesting that dysanapsis contributes to abnormal lung function in juvenile rats.

Keywords: antenatal inflammation; chronic lung disease of infancy; developmental dysanapsis; dysanapsis; neonatal lung development.

Figure 1.

Effects of antenatal endotoxin (ETX) on pup weight and crown-to-rump length at day 14 (D14). ETX decreases body weight (A) and crown–rump length (B), but does not decrease crown–rump length when corrected for body weight (C). n > 30 animals per group (A–C). **P < 0.01 and ****P < 0.0001. CTL = control; ns = no statistical significance.

Figure 2.

Effects of antenatal ETX on distal lung structure at D14. Lung histology from ETX-exposed rat pups (B) demonstrates alveolar simplification compared with control (A). Original magnification, 20×. Scale bars, 100 μm. Morphometric analysis of inflation-fixed (C and D) and perfusion-fixed (E) lung tissue demonstrates that antenatal ETX leads to distal lung simplification as quantified by decreased radial alveolar counts (C) and increased mean linear intercept (D and E) in ETX-exposed pups compared with control pups. n = 5–7 animals per group. *P < 0.05 and **P < 0.01.

Figure 3.

Stereological assessments of control and ETX-exposed rat pup lungs at D14. ETX does not lead to significantly decreased lung volume by water displacement (A), parenchymal volume (B), parenchymal air volume (C), or alveolar septal volume (D). Representative images of control (1) and ETX-exposed (2) lung parenchyma at 40× original magnification. Scale bars, 200 µm. ETX-exposed pups have decreased numbers of alveolar attachment points to airways (F), and a representative image of attachment points along the airway perimeter is shown (3). n = 6 animals per group. *P < 0.05.

Figure 4.

Effects of antenatal ETX on airway diameter at D14. Histograms demonstrate airway diameters measured by micro–computed tomography at inflation pressures of 5 cm H₂O (P5; A–C) and 20 cm H₂O (P20; D–F). (A) At P5, ETX decreases the diameters of the trachea, main bronchi (second generation), and third-generation airways. (D) At P20, only the tracheal diameter is significantly decreased in ETX-exposed pups. Representative three-dimensional micro–computed tomography images for each group (B, control P5; C, ETX P5; E, control P20; F, ETX P20). Lung histology from control (G) and ETX exposure (H) showing medium-sized airways (average diameter, 120–300 µm). Scale bars, 100 μm. There is no difference in the average airway wall thicknesses of medium (I) or large (J) airways. n = 5–10 animals per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5.

Effects of antenatal ETX on baseline lung mechanics at D14. ETX-exposed pups exhibited increased respiratory system resistance (A), decreased compliance (B), and decreased inspiratory capacity (C). ETX-exposed pups exhibited increased Newtonian resistance (D), elastance (E), and tissue damping (F). n = 15–30 animals per group. **P < 0.01 and ****P < 0.0001.

Figure 6.

Effects of antenatal ETX on airway reactivity at D14. Response to albuterol (A–C) and airway reactivity as assessed by methacholine challenge (D–F) are demonstrated. (A) ETX leads to increased respiratory system resistance (Rrs) at baseline (before albuterol). ETX-exposed pups exhibited a significant decrease in Rrs in response to albuterol. There is no statistical difference between control and ETX groups after albuterol. (B) Albuterol did not have a significant effect on airway resistance in either group. (C) ETX exposure led to decreased whole lung compliance (Crs) at baseline. The control and ETX groups exhibited increased Crs with albuterol. ETX-exposed pups had significantly lower Crs after albuterol. (D) ETX and control groups exhibited parallel Rrs responses to methacholine. In ETX-exposed and CTL pups, Rrs increased significantly from baseline at similar concentrations of methacholine. (E) ETX-exposed pups exhibited progressive dose-related increases in airway resistance with lower concentrations of methacholine. (F) Crs of ETX-exposed and control pups decreased in parallel with increasing doses of methacholine. n = 12–20 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; asterisks in A–C indicate significant differences between the compared groups; asterisks in D–F indicate significant change from baseline within an experimental group. MCH = methacholine.

Figure 7.

Effects of antenatal ETX on spirometric measures of dysanapsis at D14. Forced expiratory maneuver curves are demonstrated in control (A) and ETX-exposed (B) pups. ETX did not lead to statistical differences in FEV in 0.01 second (C) or FVC (D). ETX-exposed pups had decreased forced expiratory flow at 50% of FVC (E). ETX-exposed pups had significantly lower ratios of FEV_0.01 to FVC (F) and forced expiratory flow at 50% of FVC to FVC (G). n = 14–17 animals per group. *P < 0.05 and **P < 0.001. FEF₅₀ = forced expiratory flow at 50% of FVC.