As a result of this finding, we increased dose of TPA up to 2 mg for each nebulization and compared the results with those of the saline-treated group. Figure 2 shows the effect of TPA 2 mg nebulization on pulmonary gas exchange. PaO2/FiO2 ratio (Fig. 2A) was significantly decreased in animals nebulized with saline as compared with sham animals. Nebulization of 2 mg of TPA attenuated the fall in this variable. Statistically significant differences were observed at 30, 36, 42, and 48 h after insult. An increase in pulmonary shunt fraction (Fig. 2B) seen in the saline group was significantly attenuated by TPA 2 mg nebulization at 36 and 42 h after the combined injury.
Lung lymph flow, a characteristic of pulmonary transvascular fluid flux, was markedly increased in injured, saline nebulized animals compared with the sham group (Fig. 3). The lymph flow began to increase 12 h after the insult and a peak was observed at 42 h (7-fold increase as compared with sham group). However, nebulization of 2 mg of TPA reversed this increase in pulmonary transvascular fluid flux and significant differences were observed between the groups (saline and TPA 2 mg) at 18, 24, 30, 36, 42, and 48 h after the combined injury.
Lung bloodless wet-to-dry weight ratio, a measure of lung water content, was significantly increased at 48 h after insult in the saline group as compared with the sham group (Fig. 4). However, the nebulization of 2 mg of TPA significantly reduced this increase.
The airway obstruction score revealed a significant increase in mean obstruction of bronchi (Fig. 5) in the saline group as compared with the sham group. Treatment with 2 mg TPA significantly reduced the obstruction score.
Figure 6 shows the effect of TPA 2 mg nebulization on the increase in ventilatory pressures. The airway pressures (peak, Fig. 6A; pause, Fig. 6B) were stable in the sham group but were markedly increased in the saline group during the second one-half of the experiment, and the values were almost doubled in those animals 48 h after the insult. However, nebulization of TPA at 2 mg reduced these increases almost to normal levels.
We have previously described the pathophysiology of acute lung injury in sheep with combined burn and smoke inhalation injury (1). An increase in airway blood flow, increased pulmonary vascular permeability, airway obstruction, and pulmonary shunting are major factors that result in pulmonary dysfunction. In the present study, we report the importance of dissolution of fibrin in the maintenance of pulmonary function in the condition of thermal injury associated with smoke inhalation. In Figure 1, we showed a photograph of cast material taken from a sheep with combined burn and smoke inhalation injury. This solid mass of formed cast material nearly 30 cm long can be seen to maintain the shape of the branching airways it occupied. This material is secreted into the airway and forms a solid, gelatinous mass, narrowing or occluding the lumen. Airway obstruction caused by cast material may cause alveolar hypoxia (11) and promote bronchopneumonia. Failure to remove such obstructive cast material can create a life-threatening problem (12, 13). Thus, it is important to clear the airways to maintain adequate pulmonary or extrapulmonary organ function. Because airway obstruction is mainly caused by mucus secretion, airway epithelial cell debris, influx of inflammatory cells, and airway microvascular leakage leading to formation of a fibrin clot (4, 8, 14), pathophysiologic therapy should be designed considering those pathogenic factors. Previously, we have shown that inhibition of inducible nitric oxide synthase (iNOS)-derived excessive NO resulted in reduced airway obstruction by reducing airway blood flow in the same model (3). Using a sepsis model, our colleagues reported a beneficial effect of thrombin inhibitors, which prevent fibrin formation (8, 9). In this sepsis model induced by smoke inhalation and airway instillation of Pseudomonas aeruginosa, airways were obstructed to the same extent as in the present burn and smoke inhalation model (8, 9, 15). Soejima et al. (16) reported that smoke inhalation alone caused the acute lung injury, and the degree of tissue injury is higher in sheep with smoke inhalation alone then those with burn alone (1). Although there are no data showing the effect of smoke inhalation alone on the airway obstruction score in these studies (1, 16), significant increase of ventilatory pressures (peak and pause airway pressures) (17) could suggest that toxic smoke components could contribute to the airway obstruction. However, the combined burn and smoke inhalation induced significantly grater lung injury compared with smoke alone or burn alone groups (1). Thus, we chose the combined burn and smoke inhalation to induce the acute lung injury model in the present study.
Patients who are not immediately treated after burn and inhalation injury often develop airway casts that are hard to remove by tracheobronchial toilet. Therefore, our aim in the present study was to dissolve casts that have already formed using the fibrinolytic agent TPA. To maximally mimic a clinical situation involving delayed treatment of burn and inhalation injury, we started the nebulization of this compound 4 h after the injury. To exclude the effect of the nebulization procedure itself, we first compared the pulmonary variables in groups of injured animals without treatment or treated with nebulization of saline or TPA at 1 mg per dose. As shown in Table 1, saline itself had some positive effects on pulmonary function. The saline nebulization had a slight tendency to improve pulmonary gas exchange, but the differences were not statistically significant as compared with the control group. Nebulization of 1 mg TPA had a better effect, but still there were no statistically significant differences versus the saline group. Therefore, we increased the dose of TPA up to 2 mg and compared the effects with the saline group. The initial (1 mg) dose was chosen considering the recommendation to use the TPA (2 mg/2mL) for reestablishing the patency of occluded intravenous catheters. It was shown that doses of 1 to 2 mg of TPA effectively used for maintenance of central venous catheters (18). In addition, we successfully use TPA (0.5 mg/mL) in our laboratory for restoring of the occluded catheters inserted into lymph vessel. For this purpose, we mix the sheep plasma with TPA. The TPA mixed with sheep plasma also effectively reduced, in vitro, the size of cast material taken from sheep with combined burn and smoke inhalation injury (data not shown), suggesting that recombinant TPA might interact with sheep plasma plasminogen. This possibility should be investigated more precisely in future studies.
In the present study, the saline group of animals showed a typical cardiopulmonary response to burn/smoke injury (1, 3). The pathophysiological changes in those animals were characterized by deterioration of pulmonary gas exchange and a marked increase in pulmonary vascular permeability with formation of lung tissue edema. There was also a dramatic increase in airway obstruction associated with increased airway pressures. Although the exact mechanism remains unknown, the nebulization of the higher dose of TPA reversed these pathological changes.
Several mechanisms may exist by which airway obstruction could contribute to the pathophysiology of acute lung injury in the ovine model of combined burn and smoke inhalation injury. Near total obstruction of a few bronchi would prevent ventilation of individual lung segments (19), whereas partial obstruction would be expected to reduce ventilatory flow, producing hypoxia. Ventilation/perfusion mismatching in occluded areas can result in pulmonary shunt formation leading to poor gas exchange. On the other hand, overventilation of alveolae supplied by airways without obstruction, if mechanical ventilation is present, can cause so called ventilation-induced lung injury or barotrauma (6). In addition, overstretching of the alveolar wall activates proinflammatory cytokines such as interleukin 8 (IL-8) (7), and hypoxia itself also can modulate proinflammatory cytokines (20–22). Narimanbekov et al. (23) demonstrated that hyperventilation increased cytokine-dependent lung injury in rabbits. Cox et al. (4) demonstrated a correlation between the airway obstruction score and pulmonary gas exchange (PaO2/FiO2 ratio) in sheep with combined burn and smoke inhalation injury. The authors showed that the bronchial obstruction score was predictive of PaO2/FiO2 ratio with a correlation coefficient of 0.76. It has been reported that acute hypoxemia, sufficient to produce cyanosis, has been attributed to obstructive casts in patients with inhalation injury (14); removal of the cast was shown to resolve the critical situation and reduce airway pressures almost immediately, returning arterial oxygen tension to normal levels (13). Pruitt et al. (24) suggested that obstructive airway cast after smoke inhalation might promote atelectasis, pneumonia, and barotraumas. Taken together, the results of our present study and these previous studies suggest that airway obstruction may contribute to the acute lung injury in sheep with combined burn and smoke inhalation injury and that clearance of this obstructing cast is crucial in maintaining adequate pulmonary function. The present study suggests a potential therapeutic tool that may help to alleviate the consequences of focal airway obstruction.
Previously, we have shown the pathophysiology of acute lung injury in same model (1) and described some factors that cause lung tissue injury, including excessive nitric oxide and its toxic products such as peroxynitrite, cyclooxygenase products, proinflammatory cell accumulation, and an increase in pulmonary and bronchial microvascular permeability. Thus, we do not exclude that the above-mentioned factors participated in the pathogenesis of lung injury in this model. However, overall, evidence gathered from this study involving the use of TPA nebulization suggests that airway obstruction profoundly contributes to pulmonary function deterioration, worsening the severity of acute lung injury.
Of particular interest, alveolar and interstitial fibrin is found in the acute respiratory distress syndrome (25). An extravascular fibrin deposition characterizes most forms of acute and chronic lung injury (26). In the alveolar space, fibrin or fibrinogen can impair surfactant function (27), thereby contributing to atelectasis. Fibrin and its byproducts can influence migration of macrophages and fibroblasts (28, 29). Both fibrin and fibrinogen provide adhesion sites for inflammatory cells that are recruited to the site of tissue damage (30). Thus, fibrin could cause or worsen the lung injury in a variety of ways. Although we did not determine the fibrin deposition in this study, we do not exclude the possibility that aerosolized TPA may inhibit the fibrin or fibrinogen in alveolar or in interstitial space, thereby ameliorating the pulmonary function. This possibility should be examined in future studies. Darien et al. (31) showed that intravenous injection of heparin improved pulmonary function in porcine model of acute lung injury. Because both intravascular and intra-alveolar fibrin deposits play an important role in pathogenesis of acute respiratory distress syndrome (32, 33), the intravenous administration of TPA also should be considered in future studies.
Taken together, aerosolized TPA could be useful therapeutic agent for managing patients with thermal injury associated with smoke inhalation. Previously, we have shown beneficial effects of various aerosolized anticoagulants in prevention of airway obstruction. However, these anticoagulants have no effect on already formed fibrin clots, which are often present in patients with thermal injury, especially with delayed admission. Clinically used potent fibrinolytic agent, TPA, could be a good candidate for airway management of these patients. The reasonable cost and absence of obvious negative effects on hemostasis, if it aerosolized, raises the significance of this compound.
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Airway obstruction; lung; ARDS