Elastase-Induced Parenchymal Disruption and Airway Hyper Responsiveness in Mouse Precision Cut Lung Slices: Toward an Ex vivo COPD Model

Abstract

Background: COPD is a progressive lung disease characterized by emphysema and enhanced bronchoconstriction. Current treatments focused on bronchodilation can delay disease progression to some extent, but recovery or normalization of loss of lung function is impossible. Therefore, novel therapeutic targets are needed. The importance of the parenchyma in airway narrowing is increasingly recognized. In COPD, the parenchyma and extracellular matrix are altered, possibly affecting airway mechanics and enhancing bronchoconstriction. Our aim was to set up a comprehensive ex vivo Precision Cut Lung Slice (PCLS) model with a pathophysiology resembling that of COPD and integrate multiple readouts in order to study the relationship between parenchyma, airway functionality, and lung repair processes.Methods: Lungs of C57Bl/6J mice were sliced and treated ex vivo with elastase (2.5 μg/ml) or H2O2 (200 μM) for 16 h. Following treatment, parenchymal structure, airway narrowing, and gene expression levels of alveolar Type I and II cell repair were assessed.Results: Following elastase, but not H2O2 treatment, slices showed a significant increase in mean linear intercept (Lmi), reflective of emphysema. Only elastase-treated slices showed disorganization of elastin and collagen fibers. In addition, elastase treatment lowered both alveolar Type I and II marker expression, whereas H2O2 stimulation lowered alveolar Type I marker expression only. Furthermore, elastase-treated slices showed enhanced methacholine-induced airway narrowing as reflected by increased pEC50 (5.87 at basal vs. 6.50 after elastase treatment) and Emax values (47.96 vs. 67.30%), and impaired chloroquine-induced airway opening. The increase in pEC50 correlated with an increase in mean Lmi.Conclusion: Using this model, we show that structural disruption of elastin fibers leads to impaired alveolar repair, disruption of the parenchymal compartment, and altered airway biomechanics, enhancing airway contraction. This finding may have implications for COPD, as the amount of elastin fiber and parenchymal tissue disruption is associated with disease severity. Therefore, we suggest that PCLS can be used to model certain aspects of COPD pathophysiology and that the parenchymal tissue damage observed in COPD contributes to lung function decline by disrupting airway biomechanics. Targeting the parenchymal compartment may therefore be a promising therapeutic target in the treatment of COPD.

Cholinergic neuroplasticity in asthma driven by TrkB signaling

“…A different set of animals was used to prepare precision cut lung slices (PCLS), as described previously 34 . Briefly, following euthanasia with ketamine/domitor, the trachea was cannulated and the lungs filled with a buffered 1.5% of low melting‐point agarose solution.…”

“…PCLSs were generated at a thickness of 250 μm. After washing, they were incubated in Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with sodium pyruvate (1 mM), nonessential amino acid mixture (1:100), gentamycin (45 mg/mL), penicillin (100 U/mL), streptomycin (100 mg/mL), and amphotericin B (1.5 mg/mL) at 37°C in a humidified chamber under 5% of CO2/95%, as described previously 34,35 …”

“…Twenty four hours after incubation, the PCLS were used for airway narrowing studies using methacholine or EFS. Briefly, methacholine dose response curves (10‐9 M‐10‐2 M) were recorded by placing the slice in 1 mL medium and fixing it with a nylon mesh and metal washer as described previously 34,36 . For EFS measurements, the slices were inserted in 2 mL medium between two platinum electrodes, which generate increasing frequencies of electric pulses.…”

OptiJ: Open-source optical projection tomography of large organ samples

“…Furthermore, we implemented for the first time Fourier Ring Correlation (FRC) as a resolution measure for reconstructed OPT data sets. The non-destructive 3D imaging of COPD mice models lung lobes could provide a whole-organ perspective of alveolar cell clusters in an intact lung, where the involvement of specific cell types in pathophysiological processes could be tracked and quantified, complementary to recent studies of COPD pathophysiology with confocal microscopy (58). Although immunostaining with the anti-surfactant protein C was only partially successful, we were able to make use of autofluorescence from elastin and collagen in epithelial cells and extracellular matrix from the large airway wall to boost signals and obtain high-contrast images of the large airway tree.…”

The Novel TRPA1 Antagonist BI01305834 Inhibits Ovalbumin-Induced Bronchoconstriction in Guinea Pigs

“…Using a microscope (Eclipse, TS100; Nikon) time-lapse images (1 frame per 2 s) of the lung slices were captured. The airway luminal area was quanti ed using image acquisition software (NIS-elements; Nikon) and expressed as percentage of basal area, as described previously (Maarsingh et al, 2019, Van Dijk et al, 2017.…”