Preexposure to hyperoxia causes increased lung injury and epithelial apoptosis in mice ventilated with high tidal volumes

Abstract

Both high tidal volume mechanical ventilation (HV) and hyperoxia (HO) have been implicated in ventilator-induced lung injury. However, patients with acute lung injury are often exposed to HO before the application of mechanical ventilation. The potential priming of the lungs for subsequent injury by exposure to HO has not been extensively studied. We provide evidence that HO (90%) for 12 h followed by HV (25 μl/g) combined with HO for 2 or 4 h (HO-12h+HVHO-2h or -4h) induced severe lung injury in mice. Analysis of lung homogenates showed that lung injury was associated with cleavage of executioner caspases, caspases-3 and -7, and their downstream substrate poly(ADP-ribose) polymerase-1 (PARP-1). No significant lung injury or caspase cleavage was seen with either HO for 16 h or HV for up to 4 h. Ventilation for 4 h with HO (HVHO) did not cause significant lung injury without preexposure to HO. Twelve-hour HO followed by lower tidal volume (6 μl/g) mechanical ventilation failed to produce significant injury or caspase cleavage. We also evaluated the initiator caspases, caspases-8 and -9, to determine whether the death receptor or mitochondrial-mediated pathways were involved. Caspase-9 cleavage was observed in HO-12h+HVHO-2h and -4h as well as HO for 16 h. Caspase-8 activation was observed only in HO-12h+HVHO-4h, indicating the involvement of both pathways. Immunohistochemistry and in vitro stretch studies showed caspase cleavage in alveolar epithelial cells. In conclusion, preexposure to HO followed by HV produced severe lung injury associated with alveolar epithelial cell apoptosis.

Fig. 1.

Preexposure to hyperoxia (HO) followed by high tidal volume mechanical ventilation (HV) with HO caused lung injury. n = 4 Mice per group. Hematoxylin-eosin (H&E)-stained, formalin-fixed, paraffin-embedded lung tissues from mice, subjected to spontaneously breathing with normoxia (SVNO; A), HV with 2-h normoxia (HVNO-2h; B), HVNO-4h (C), SVHO-4h (D), SVHO-16h (E), HVHO-4h (F), HO for 12 h followed by HV combined with HO for 2 h (12h-HO+HVHO-2h; G), 12h-HO+HVHO-4h (H), low tidal volume ventilation with HO for 4 h after 12-h preexposure to HO (12h-HO+LVHO-4h; I), 12h-HO+HVHO-2h (G), and 12h-HO+HVHO-4h (H) conditions show evidence of extensive lung injury with interstitial and alveolar edema (↑), hemorrhage (←), and acute inflammatory cell infiltration (▲) compared with other experimental conditions (scale: 100 μm).

Fig. 2.

The composite lung injury scores (LIS; left) represent the sum of the mean injury subtype scores for each condition on a scale of 0–16 (n = 4 mice per group). The LIS were significantly higher in animals preexposed to HO for 12 h followed by 2 and 4 h of HV and HO (P < 0.05). No other condition showed statistically significant increases in lung injury compared with controls (SVNO). *P < 0.05 compared with SVNO. Bronchoalveolar lavage fluid (BALF) protein concentrations (right) mirror the LIS (n = 3 mice per group).

Fig. 3.

Quasi-static thoracic compliance for selected experimental conditions at time 0 and at the end of the experiment. All measurements remained stable except in the 12h-HO+HVHO-2h and 12h-HO+HVHO-4h conditions where the compliance decreased. *P < 0.05 compared with same condition at time 0 and with SVNO.

Fig. 4.

A: activation of executioner caspases (caspases-3 and -7) and their downstream substrate, poly(ADP-ribose) polymerase-1 (PARP-1), in whole lung homogenates of mice subjected to SVNO (lane 1), HVNO-2h (lane 2), HVNO-4h (lane 3), SVHO-4h (lane 4), SVHO-16h (lane 5), HVHO-4h (lane 6), 12h-HO+HVHO-2h (lane 7), 12h-HO+HVHO-4h (lane 8), and 12h-HO+LVHO-4h (lane 9). B, C, and D show densitometry for caspase-3 (Casp-3), caspase-7, and PARP-1, respectively. Caspase and PARP-1 cleavage were observed only in 12h-HO+HVHO-2h and -4h. No significant caspase cleavage was observed in any other condition. Blots shown are representative from 3 different animals per group. *P < 0.05 compared with SVNO.

Fig. 5.

A: activation of initiator caspases (caspases-8 and -9) in whole lung homogenates of mice subjected to SVNO (lane 1), HVNO-2h (lane 2), HVNO-4h (lane 3), SVHO-4h (lane 4), SVHO-16h (lane 5), HVHO-4h (lane 6), 12h-HO+HVHO-2h (lane 7), 12h-HO+HVHO-4h (lane 8), and 12h-HO+LVHO-4h (lane 9). B and C show densitometry for caspase-8 and caspase-9, respectively. Both the initiator caspases were activated in 12h-HO+HVHO-2h and -4h conditions, whereas an increase in cleaved caspase-9 was observed in SVHO-16h. No other conditions produced cleavage of either initiator caspase. Blots shown are representative from 3 different animals per group. *P < 0.05 compared with SVNO.

Fig. 6.

Immunohistochemistry of cleaved caspase-3 in control SVNO mice (A) and mice exposed to 12h-HO+HVHO-4h (B). Confocal imaging of immunofluorescence staining for cleaved PARP-1 in mouse lungs subjected to SVNO (C) and to 12h-HO+HVHO-4h (D). SVNO produced no significant positive staining for either cleaved caspase-3 or cleaved PARP-1, whereas epithelial cells stained positive for both cleaved caspase-3 and cleaved PARP-1 antibodies after exposure to 12h-HO+HVHO-4h (n = 3 animals per group). Representative images from a single animal are shown in 100-μm scale.

Fig. 7.

Activation of executioner caspases, caspases-3 and -7, and their downstream substrate, PARP-1, in a mouse alveolar epithelial cell line (MLE-12) subjected to SVNO (static normoxia; lane 1), HVNO-4h (20% cyclic stretch normoxia; lane 2), HVHO-4h (20% cyclic stretch HO; lane 3), SVHO-16h (static HO; lane 4), 12h-HO+HVHO-4h (12-h static HO followed by 20% cyclic stretch HO; lane 5), and 12h-HO+LVHO-4h (12-h static HO followed by 6% cyclic stretch HO; lane 6) in Fig. 6, A–C. Increased caspase-3/7 cleavage was observed after 12h-HO+HVHO-4h (*P < 0.05), whereas significant PARP-1 cleavage was seen in HVNO-4h and 12h-HO+HVHO-4h conditions (*P < 0.05). All 3 blots from each set of experiments were developed on the same film. The blotting, development, and exposure conditions were the same for all 3 independent experiments. Representative blots are shown above the densitometry data from 3 independent experiments in which 4 wells were pooled for each condition.