Hyperoxia provokes a time- and dose-dependent inflammatory response in mechanically ventilated mice, irrespective of tidal volumes

Abstract

Background: Mechanical ventilation and hyperoxia have the potential to independently promote lung injury and inflammation. Our purpose was to study both time- and dose-dependent effects of supplemental oxygen in an experimental model of mechanically ventilated mice.

Methods: Healthy male C57Bl/6J mice, aged 9-10 weeks, were intraperitoneally anesthetized and randomly assigned to the mechanically ventilated group or the control group. In total, 100 mice were tracheotomized and mechanically ventilated for either 8 or 12 h after allocation to different settings for the applied fractions of inspired oxygen (FiO₂, 30, 50, or 90%) and tidal volumes (7.5 or 15 ml/kg). After euthanisation arterial blood, bronchoalveolar lavage fluid (BALf) and tissues were collected for analyses.

Results: Mechanical ventilation significantly increased the lung injury score (P < 0.05), mean protein content (P < 0.001), and the mean number of cells (P < 0.01), including neutrophils in BALf (P < 0.001). In mice ventilated for 12 h, a significant increase in TNF-α, IFN-γ, IL-1β, IL-10, and MCP-1 (P < 0.01) was observed with 90% FiO₂, whereas IL-6 showed a decreasing trend (P for trend = 0.03) across FiO₂ groups. KC, MIP-2, and sRAGE were similar between FiO₂ groups. HMGB-1 was significantly higher in BALf of mechanically ventilated mice compared to controls and showed a gradual increase in expression with increasing FiO₂. Cytokine and chemokine levels in BALf did not markedly differ between FiO₂ groups after 8 h of ventilation. Differences between the tidal volume groups were small and did not appear to significantly interact with the oxygen levels.

Conclusions: We demonstrated a severe vascular leakage and a pro-inflammatory pulmonary response in mechanically ventilated mice, which was enhanced by severe hyperoxia and longer duration of mechanical ventilation. Prolonged ventilation with high oxygen concentrations induced a time-dependent immune response characterized by elevated levels of neutrophils, cytokines, and chemokines in the pulmonary compartment.

Keywords: Hyperoxia; Inflammation; Mechanical ventilation; Oxygen toxicity; VILI; mice.

Fig. 1

Arterial oxygenation and ventilation parameters. Data are means ± SEM. Arterial oxygenation in carotid blood (a, upper left panel), arterial carbon dioxide in carotid blood (b, upper right panel), PaO₂/FiO₂ ratio (c, lower left panel), and dynamic compliance (d, lower right panel). Facets within the panels represent mechanical ventilation time. Different colors represent different tidal volume groups, and different transparency levels represent different FiO₂ groups. 0h no mechanical ventilation time, control group; 8h 8 h of mechanical ventilation, 12h 12 h of mechanical ventilation, LTV low tidal volumes, HTV high tidal volumes. Dynamic lung compliance (tidal volume size/(peak inspiratory pressure − PEEP) was measured hourly. PaO₂ and PaCO₂ were measured once in the arterial blood gas sample taken from the carotid artery at the end of the experiment

Fig. 2

Markers of lung injury in BALf after indicated study interval. Data are means ± SEM. Lung injury score (a, upper left panel), total protein content (b, upper right panel), total cell counts (c, lower left panel), and proportion of neutrophils (d, lower right panel) in BALf obtained after the study interval. Facets within the panels represent mechanical ventilation time. Different colors represent different tidal volume groups, and different transparency levels represent different FiO₂ groups. 0h no mechanical ventilation time, control group; 8h 8 h of mechanical ventilation, 12h 12 h of mechanical ventilation, LTV low tidal volumes, HTV high tidal volumes

Fig. 3

Inflammatory mediators in BALf in controls and after 12 h of mechanical ventilation. Cytokine and chemokine levels in BALf obtained after 12 h of mechanical ventilation. Data are means ± SEM. Facets within the panels represent mechanical ventilation time. Different colors represent different tidal volume groups, and different transparency levels represent different FiO₂ groups. 0h no mechanical ventilation time, control group; 12h 12 h of mechanical ventilation, LTV low tidal volumes, HTV high tidal volumes. Cuzick’s P for trend in increasing oxygen levels at 12 h of mechanical ventilation: IL-1β, <0.001; MIP-2, 0.27; IL-6, 0.03; KC, 0.22; IL-10, 0.001; MCP-1, <0.001; TNF-α, 0.001; IFN-γ, 0.001; sRAGE, 0.11; HMGB-1, 0.001. *P for trend <0.05; **P for trend <0.01; ***P for trend <0.001