Nebulization with γ-tocopherol ameliorates acute lung injury after burn and smoke inhalation in the ovine model

Abstract

We hypothesize that the nebulization of γ-tocopherol (g-T) in the airway of our ovine model of acute respiratory distress syndrome will effectively improve pulmonary function following burn and smoke inhalation after 96 h. Adult ewes (n = 14) were subjected to 40% total body surface area burn and were insufflated with 48 breaths of cotton smoke under deep anesthesia, in a double-blind comparative study. A customized aerosolization device continuously delivered g-T in ethanol with each breath from 3 to 48 h after the injury (g-T group, n = 6), whereas the control group (n = 5) was nebulized with only ethanol. Animals were weaned from the ventilator when possible. All animals were killed after 96 h, with the exception of one untreated animal that was killed after 64 h. Lung g-T concentration significantly increased after g-T nebulization compared with the control group (38.5 ± 16.8 vs. 0.39 ± 0.46 nmol/g, P < 0.01). The PaO(2)/FIO(2) ratio was significantly higher after treatment with g-T compared with the control group (310 ± 152 vs. 150 ± 27.0, P < 0.05). The following clinical parameters were improved with g-T treatment: pulmonary shunt fraction, peak and pause pressures, lung bloodless wet-to-dry weight ratios (2.9 ± 0.87 vs. 4.6 ± 1.4, P < 0.05), and bronchiolar obstruction (2.0% ± 1.1% vs. 4.6% ± 1.7%, P < 0.05). Nebulization of g-T, carried by ethanol, improved pulmonary oxygenation and markedly reduced the time necessary for assisted ventilation in burn- and smoke-injured sheep. Delivery of g-T into the lungs may be a safe, novel, and efficient approach for management of acute lung injury patients who have sustained oxidative damage to the airway.

Figure 1

Protocol for Weaning from Mechanical Ventilation. Weaning from ventilator was initiated if PaO₂/FiO₂ ratio is above 250 at 48 or more hours after injury.

Figure 2

Gamma- and Alpha-Tocopherol Concentrations after Burn and Smoke Inhalation Injury with and without Gamma-Tocopherol Nebulization. After a 40% TBSA full thickness burn and a smoke inhalation injury, sheep were killed after 96 hours and lung homogenate was used to indirectly measure oxidative stress. Results were compared to injured animals that were nebulized with vitamin E. Oxidative stress was evaluated by measuring ROS and RNS scavenger gamma-tocopherol in the (A) lung and (C) liver, and ROS scavenger alpha-tocopherol in the (B) lung and (D) liver. Lung oxidative stress was attenuated post-injury with vitamin E treatment based on the associated increases in tocopherols. Data are shown as means ± SEM. *p < 0.05 versus injured animals sacrificed after 96 hours.

Figure 2

Gamma- and Alpha-Tocopherol Concentrations after Burn and Smoke Inhalation Injury with and without Gamma-Tocopherol Nebulization. After a 40% TBSA full thickness burn and a smoke inhalation injury, sheep were killed after 96 hours and lung homogenate was used to indirectly measure oxidative stress. Results were compared to injured animals that were nebulized with vitamin E. Oxidative stress was evaluated by measuring ROS and RNS scavenger gamma-tocopherol in the (A) lung and (C) liver, and ROS scavenger alpha-tocopherol in the (B) lung and (D) liver. Lung oxidative stress was attenuated post-injury with vitamin E treatment based on the associated increases in tocopherols. Data are shown as means ± SEM. *p < 0.05 versus injured animals sacrificed after 96 hours.

Figure 3

Pulmonary Gas Exchange after Burn and Smoke Inhalation Injury with and without Gamma-Tocopherol Nebulization. After a 40% TBSA full thickness burn and a smoke inhalation injury, sheep were killed after 96 hours. Results were compared to injured animals that were nebulized with vitamin E. Pulmonary gas exchange was evaluated by measuring (A) PaO₂/FiO₂, and (B) pulmonary shunt fraction. Pulmonary gas exchange significantly improved with vitamin E treatment. Data are shown as means ± SEM. * P < 0.05 versus injured animals sacrificed after 96 hours.

Figure 3

Pulmonary Gas Exchange after Burn and Smoke Inhalation Injury with and without Gamma-Tocopherol Nebulization. After a 40% TBSA full thickness burn and a smoke inhalation injury, sheep were killed after 96 hours. Results were compared to injured animals that were nebulized with vitamin E. Pulmonary gas exchange was evaluated by measuring (A) PaO₂/FiO₂, and (B) pulmonary shunt fraction. Pulmonary gas exchange significantly improved with vitamin E treatment. Data are shown as means ± SEM. * P < 0.05 versus injured animals sacrificed after 96 hours.

Figure 4

Peak and Pause Pressures after Burn and Smoke Inhalation Injury with and without Gamma-Tocopherol. After a 40% TBSA full thickness burn and a smoke inhalation injury, sheep were killed after 96 hours. Results were compared to injured animals that were nebulized with vitamin E. Compliance was evaluated by measuring (A) peak pressure, and (B) pause pressure in the first 48 hours after injury. Peak and pause pressure both significantly decreased and improved with vitamin E treatment after 42 and 48 hours. Data are shown as means ± SEM. * P < 0.05 versus injured animals sacrificed after 96 hours.

Figure 5

Obstruction Scores of Bronchi and Bronchioles and Lung Wet-to-Dry Ratio after Burn and Smoke Inhalation Injury with and without Gamma-Tocopherol. After a 40% TBSA full thickness burn and a smoke inhalation injury, sheep were killed after 96 hours. Results were compared to injured animals that were nebulized with vitamin E. Pulmonary pathophysiology was evaluated by measuring (A) bloodless wet-to-dry ratio, and (B) obstruction score. Both obstruction of the bronchioles and bronchi and wet-to-dry ratio significantly decreased and improved with vitamin E treatment after 42 and 48 hours. Data are shown as means ± SEM. *p < 0.05 versus injured animals sacrificed after 96 hours.

Figure 6

Poly (ADP Ribose)-Polymerase (PAR) Activation Significantly Decreases with vitamin E Treatment after Burn and Smoke Inhalation Injury. After a 40% TBSA full thickness burn and a smoke inhalation injury, sheep were killed after 96 hours. Representative Western blot analysis of the (A) poly(ADP-ribosylated) (PAR) proteins indicating the PARP activity in sheep lung. The poly(ADP-ribosylation) of the proteins was significantly decreased by 96 hours with vitamin E treatment after burn and smoke inhalation. Equality of protein loading was confirmed by the expression of β-actin. (B) Densitometric evaluation of PAR/actin ratios. Data are shown as means ± SEM. *p<0.05 vs. injured animals.