Time course of inflammation, oxidative stress and tissue damage induced by hyperoxia in mouse lungs

Abstract

In this study our aim was to investigate the time courses of inflammation, oxidative stress and tissue damage after hyperoxia in the mouse lung. Groups of BALB/c mice were exposed to 100% oxygen in a chamber for 12, 24 or 48 h. The controls were subjected to normoxia. The results showed that IL-6 increased progressively after 12 (P < 0.001) and 24 h (P < 0.001) of hyperoxia with a reduction at 48 h (P < 0.01), whereas TNF-α increased after 24 (P < 0.001) and 48 h (P < 0.001). The number of macrophages increased after 24 h (P < 0.001), whereas the number of neutrophils increased after 24 h (P < 0.01) and 48 h (P < 0.001). Superoxide dismutase activity decreased in all groups exposed to hyperoxia (P < 0.01). Catalase activity increased only at 48 h (P < 0.001). The reduced glutathione/oxidized glutathione ratio decreased after 12 h (P < 0.01) and 24 h (P < 0.05). Histological evidence of lung injury was observed at 24 and 48 h. This study shows that hyperoxia initially causes an inflammatory response at 12 h, resulting in inflammation associated with the oxidative response at 24 h and culminating in histological damage at 48 h. Knowledge of the time course of inflammation and oxidative stress prior to histological evidence of acute lung injury can improve the safety of oxygen therapy in patients.

Figure 1

The cytotoxicity was evaluated using the MTT assay with the bronchoalveolar lavage cells cultured in DMEM. After incubation, the absorbance was measured at 540 nm. The cytotoxicity is represented by a black circle after 12, 24 and 48 h of hyperoxia or with a white circle after normoxia. The values are expressed as percentages relative to the control group by the mean ± standard error of the mean (SEM). The groups were tested for significance using the Kruskal–Wallis test followed by the Dunn post hoc test; the significance level was set as 5%. *P < 0.05 compared with the control group (N = 5 per group). This test was performed in the first experiment only.

Figure 2

MMP-2 and MMP-9 activities in the lung tissue homogenates from mice exposed to normoxia (control group) or hyperoxia (12, 24 and 48 h). The samples (30 μg protein) were subjected to electrophoresis in a separating gel containing gelatin. After electrophoresis, the gel was stained with Coomassie blue, and the MMP-2 and/or MMP-9 activities appeared as clear bands against the blue background. The positive control was human placenta. The negative bands were assessed using densitometry (N = 2 per group). This test was performed in the first experiment only.

Figure 3

Western blotting for IL-6 expression in the lung tissue homogenates from mice exposed to normoxia (control group) or hyperoxia (12, 24 and 48 h). The samples were subjected to electrophoresis through a separating gel. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes, and the specific primary antibody for IL-6 was incubated with the membrane overnight. After incubation with the secondary antibody, DAB was used to detect the immunoreactive bands. This procedure was repeated two times. The membrane was stained with Ponceau red as the control (N = 1 per group). This test was performed in the first experiment only.

Figure 4

Lung histology in sections stained with haematoxylin and eosin. (a) Control mouse exposed to normoxia with alveolar septa and pulmonary capillaries preserved. (b) Mouse exposed to hyperoxia for 12 h with alveolar septa and pulmonary capillaries preserved, but with interstitial cellularity increased. (c) Mouse exposed to hyperoxia for 24 h with few alveolar macrophages and neutrophils. (d) Mouse exposed to hyperoxia for 48 h with alveolar macrophages and neutrophils. These images are representative of all mice analysed; N = 10 for all experiments. The arrows indicate leucocytes (alveolar macrophages or neutrophils). This test was performed in the first experiment only.

Figure 5

Lung histology in sections immunostained for TNF-α. (a) Lung parenchyma from a control mouse exposed to normoxia with reduced TNF-α expression. (b) Lung parenchyma from a mouse exposed to hyperoxia for 12 h with reduced TNF-α expression. (c) Lung parenchyma from a mouse exposed to hyperoxia for 24 h with a high TNF-α expression level, mainly in alveolar cells. (d) Lung parenchyma from a mouse exposed to hyperoxia for 48 h, with cells expressing TNF-α. These images are representative of all mice analysed; N = 10 for all experiments. The arrows indicate leucocytes (alveolar macrophages or neutrophils) expressing TNF-α. This test was performed in the first experiment only.

Figure 6

Lung histology in sections immunostained for TNF-α. (a) Bronchi from a control mouse exposed to normoxia with bronchial cells expressing TNF-α. (b) Bronchi from a mouse exposed to hyperoxia for 12 h with bronchial cells expressing TNF-α. (c) Bronchi from a mouse exposed to hyperoxia for 24 h with reduced TNF-α expression. (d) Bronchi from a mouse exposed to hyperoxia for 48 h with reduced TNF-α expression. These images are representative of all mice analysed; N = 10 for all experiments. This test was performed in the first experiment only.