In a previous study by our laboratory, the nebulization of g-T attenuated burn and smoke inhalation-induced pathological changes and improved pulmonary function after 48 hours (18
). In our current study, we have nebulized a larger concentration of g-T and analyzed additional variables in the 96-hour late and recovery stage of burn and smoke inhalation such as ventilator weaning, peak and pause pressures, and shunt fraction. Nebulization with gamma-tocopherol into the lungs of sheep with a burn and smoke inhalation injury ameliorated the damage observed in those nebulized with the vehicle. Specifically, g-T-treatment facilitated more effective pulmonary gas exchange (PaO2
and pulmonary shunt fraction) and led to less obstruction in the bronchi and bronchioles of the lung, and less edema in the lungs (bloodless wet-to-dry ratio). We hypothesized that pulmonary changes associated with the acute phase of burn and smoke inhalation injury are caused by increased oxidative and nitrosative stress. We tested our hypothesis by delivering gamma-tocopherol into the airway to act as an anti-oxidant, scavenge ROS and RNS, and attenuate pulmonary pathophysiology after combined burn and smoke inhalation injury.
We have reported that NO generated from inducible nitric oxide synthase (NOS) has an important role in the changes in both systemic and pulmonary microvascular permeability, which follow combined burn and smoke inhalation injury (9
). Burn and smoke inhalation injury is associated with a systemic inflammatory response, and increased levels of RNS and ROS in the lung (13
). Peroxynitrite is a strong oxidant, and a nitrating and nitrosating agent, that can readily trigger DNA single-strand breakage and induce poly (ADP-ribose) polymerase (PARP) activation (32
). Intracellular NAD and ATP levels are depleted as a consequence of PARP activation (46
). In addition, PARP has been shown to be involved in the regulation of inflammatory processes, being functionally associated with nuclear factor-κB (NF-κB) (40
). g-T and a-T are scavengers of RNS and ROS. Although g-T and a-T are both potent lipophilic antioxidants, g-T has a unique function. g-T is a more effective RNS scavenger than a-T because it has an unsubstituted 5-position on the chromanol ring (10
Lipid peroxidation markers have been measured by our group and have significantly increased in survivors of burn injury compared to nonsurvivors. Malondialdehyde, which is a large mutagenic ROS, has been previously measured in sheep with burn and smoke inhalation injury with and without vitamin E. It significantly increased in sheep without vitamin E treatment. In our present study, lung oxidative stress was indirectly measured by analyzing g-T and a-T concentrations by HPLC. Lung g-T and a-T concentrations significantly increased after the nebulization of vitamin E (Figure 2.1). The plasma a-T and g-T concentrations did not change dramatically because the nebulized E does not cross into the circulation (data not shown). The lung data illustrates that our novel lipid nebulization device that was used in the present study can aerosolize viscous lipid materials effectively. It creates 2.5–5.0 μm droplets of vitamin E and is synchronized with the ventilator to deliver it only during the inspiratory cycle (18
A previous study from our laboratory showed that the increases in oxidative and nitrosative stress markers were significantly decreased in injured sheep treated with a mixed a-T and g-T solution and sacrificed after 48 hours (610 mg/g of g-T and 91 mg/g of a-T nebulized over 48 hours) (9
). In the present study, we nebulized a larger amount of g-T (950 mg/g of g-T and 40 mg/g of a-T) from 3 to 48 hours after injury and sacrificed the animals after 96 hours. The larger concentration of g-T attenuated the changes not only during the subsequent 48 h period but also the late and recovery stage after 96 hours. Additional differences between the present and previously reported studies are that all of the g-T treated animals could be weaned from the ventilator, while none of the control animals were weaned (), and the shunt fraction and peak and pause pressures were significantly improved (, ).
Another difference between our previously reported study (18
) and the present study is that ethanol was used as the vehicle carrier instead of flaxseed oil. 100% g-T is a viscous lipid material and difficult to aerosolize using our novel lipid nebulization without a carrier. Ethanol has been reported as a low-toxicity solvent for inhalation delivery (33
) and has been used in nebulized and metered dose inhalers and formulations for human use (4
). Therefore, we selected ethanol as a vehicle for nebulization with g-T. There are reports that nebulization with ethanol reduces the pathophysiology of pulmonary edema perhaps through its effects as an anti-foam agent (2
). Sisson et.al.
reported that brief exposure to a mild concentration of ethanol may enhance mucociliary clearance, stimulate bronchodilation, and attenuate the airway inflammation and injury observed in asthma and chronic obstructive pulmonary dysfunction (COPD) (33
). Furthermore, Oldenburg et al.
reported that brief ethanol exposure prevents methacholine-stimulated rat airway smooth muscle cell contraction in vivo (26
). Although tocopherol was very effective in ablating many of the pathophysiological changes noted with inhalation injury, the control group that received ethanol alone appeared to have less of an injury in comparison to our past studies. Our previous findings in the sheep model of burn and smoke inhalation have shown that 1.1 mL ethanol nebulized into the lung over a 24 hour period did not worsen lung function or mortality (which is usually 60–75%) (14
). Additionally, in our previous study (18
), g-T improved wet-to-dry weight ratios and obstruction scores significantly compared to the saline control, but not compared to the appropriate flaxseed oil control. The flaxseed oil itself restored g-T levels to baseline values. Thus, the omission of the flaxseed oil carrier allows the extent of the significant differences from the control group to be observed much more clearly.
Gamma-tocopherol nebulization significantly reduced the wet/dry weight ratio in lung tissue (). Nebulization with the g-T solution for the first 48 h increased lung g-T levels at 96 h post-injury more than 100-fold. a-T concentrations were also significantly higher in the g-T group as compared to the control group (). Since the g-T contains some a-T, it is unclear whether the increase was due to the administration of the solution, or whether the supply of g-T protected endogenous a-T from oxidation. The g-T nebulization decreased severe signs of acute lung injury as evidenced by deteriorated pulmonary gas change, massive airway obstruction, pulmonary edema, and PARP activation at 96 h post-injury. It was difficult to compare pulmonary function evidenced by Pa2
ratio in each group after 48 h post-injury, because only control animals were supported by mechanical ventilation between 48 and 96 h after injury. Therefore, the weaning process was compared between groups to show the effects of treatment in the recovery stage. The weaning could be initiated significantly earlier in the g-T group after 48 h post injury compared to the control group. The tracheostomy tube of all g-T nebulization animals could be removed within 96 h post injury, whereas none of the control animals could be completely weaned nor their tracheostomy tubes removed. In human intensive care units, prolonged intubation is known to be associated with ventilator-induced lung injury, ventilator-associated pneumonia, patient discomfort, and the need for high dose sedation (3
). Quicker weaning from the mechanical ventilator and removal of the endotracheal tube are expected to reduce these problems.
In summary, we report that burn and smoke inhalation injury significantly reduces lung alpha- and gamma-tocopherol concentrations and increases markers of pulmonary pathophysiology such as deteriorated pulmonary gas exchange, increased peak and pause pressures, massive airway obstruction, and increased pulmonary edema, while nebulization with gamma-tocopherol attenuated the injury, improved pulmonary oxygenation, and markedly reduced ventilator time. Pulmonary gamma-tocopherol delivery in ethanol may be a safe, novel, and effective treatment of patients with the acute phase of burn and smoke inhalation.