Extensive Analysis of Elastase-Induced Pulmonary Emphysema in Rats: ALP in the Lung, a New Biomarker for Disease Progression?

Abstract

It is accepted that pulmonary exposure of rodents to porcine pancreatic elastase (ELT) induces lesions that morphologically resemble human emphysema. Nonetheless, extensive analysis of this model has rarely been conducted. The present study was designed to extensively examine the effects of ELT on lung inflammation, cell damage, emphysematous change, and cholinergic reactivity in rats. Intratracheal administration of two doses of ELT induced 1) a proinflammatory response in the lung that was characterized by significant infiltration of macrophages and an increased level of interleukin-1beta in lung homogenates, 2) lung cell damage as indicated by higher levels of total protein, lactate dehydrogenase, and alkaline phosphatase (ALP) in lung homogenates, 3) emphysema-related morphological changes including airspace enlargement and progressive destruction of alveolar wall structures, and 4) airway responsiveness to methacholine including an augmented Rn value. In addition, ELT at a high dose was more effective than that at a low dose. This is the novel study to extensively analyze ELT-induced lung emphysema, and the analysis might be applied to future investigations that evaluate new therapeutic agents or risk factors for pulmonary emphysema. In particular, ALP in lung homogenates might be a new biomarker for the disease progression/exacerbation.

Keywords: ALP; LDH; airway hyperresponsiveness; pulmonary emphysema; rat.

Fig. 1

Cellularity in BALF after intratracheal challenge. BALF from each rat were collected 21 days after the intratracheal administration of ELT. Total cell counts were performed using a Berker-Tulk counting chamber. Differential cell counts were assessed in cytologic preparations stained with Diff-Quik. The results are means ± SEM (n = 6–8 in each group). (a) total cells, (b) macrophages, (c) neutrophils, (d) monocytes. **p<0.01 vs the sham group.

Fig. 2

Cytokine and chemokine profiles in the lung after intratracheal challenge. Lung tissue samples of each rat were harvested 21 days after the intratracheal administration of ELT. The levels of IL-1β (a), IL-4 (b), IL-6 (c), IL-13 (d), CINC-1 (e), and MCP-1 (f) in the lung tissue supernatants were measured by ELISA. The results are means ± SEM (n = 6–8 in each group). **p<0.01 vs the sham group.

Fig. 3

The levels of total protein, LDH, γ-GTP, and ALP in the lung after intratracheal challenge with ELT. The lung tissue samples of each rat were harvested 21 days after the intratracheal administration of ELT. The levels of total protein (a) LDH (b), γ-GTP (c), and ALP (d) in the lung tissue supernatants were measured. The results are means ± SEM (n = 6–8 in each group). *p<0.05 versus the sham group, **p<0.01 vs the sham group.

Fig. 4

Microscopic findings in the lung after intratracheal challenge with ELT. Histologic changes in the lung of each rat were evaluated 21 days after the intratracheal administration of ELT. The sections were stained with hematoxylin and eosin, and histologic analyses were performed using a microscope (bar: 100 µm). (a) Sham group, (b) ELT 20 U group, (c) ELT 160 U group.

Fig. 5

Dose response curve of inhaled MCh after intratracheal challenge with ELT. Airway responsiveness/lung function to MCh, were measured by a flexiVent 21 days after the intratracheal administration of ELT. The parameters of R (a), E (b), and C (c) were measured by fitting the linear single-compartment model (using the snapshot method). The parameters of Rn (d), G (e), and H (f) were measured by fitting the constant phase model (using the FOT method). Data are shown as means ± SEM (n = 10–12 in each group).