Our study demonstrated that airway casts produced after inhalation of the SM analog CEES were composed of abundant fibrin, a plasma-derived protein, and that the casts were consistently found only within the proximal half of the conducting airways (generations 2–15 from the total of 25 airway generations microdissected routinely). In addition, we showed that other plasma-derived proteins such as IgM also appeared within the airway lumen, indicating the presence of an early vascular insult leading to increased permeability. With the use of monastral blue pigment, we identified the bronchial circulation to be directly involved in the vascular leakage and, thereby, formation of casts. Injured bronchial vessels caused extravasation of plasma components into adjacent regions, which then appeared within airways. Once within airways, plasma components organized into casts capable of causing obstruction and compromising respiratory function.
Obstructive cast formation, often referred to as plastic bronchitis or cast bronchitis, is not unique to sulfur mustard exposure. Airway casts have been found in children with congenital heart diseases particularly after Fontan procedures (30
), as well as in patients with asthma (32
), cystic fibrosis (35
), sickle cell disease with acute chest syndrome (36
), and allergic and infectious pulmonary states (37
), and after burns or smoke inhalation injury (38
). Although plastic bronchitis is not common, it is commonly fatal (44
). Mortality rates reported for congenital heart disease–associated casts is 15–50% because of complete airway occlusion (45
). For casts due to inhalation burn injury, mortality reaches 20–30% (43
). Reports of fatal plastic bronchitis in children with asthma have also been published (32
), but with lesser frequency. The composition of casts (fibrin vs. mucin) has been used as a guide to their prognosis and treatment. The Seear classification system (30
) was developed to classify casts into two types: type I (fibrin casts with abundant inflammatory cells, particularly eosinophils) and type II (mucin casts with minimal cellularity). The presence of type I, or predominantly fibrin casts, results in a more ominous course, as seen with congenital heart disease, inhalational burns, and also in asthma. In our model, casts contained abundant fibrin. Although some airway epithelial cells and scant inflammatory cells were present in the “early casts” at 18 hours, no eosinophils were found. By 72 hours, increased numbers of neutrophils appeared, with the addition of newly deposited collagen and with the appearance of spindle cells (fibroblasts or myofibroblasts). A potential explanation for the relative paucity of inflammatory cells within the early casts in our model was that 18 hours may be an insufficient amount of time for an extensive inflammatory response to be fully manifested within the airway lumen. Indeed, analysis of BALF at 18 hours revealed an only minimal increase in absolute number of neutrophils over ethanol, further demonstrating a present but subdued inflammatory response within the airways. Whereas casts from asthma, congenital heart disease, or cystic fibrosis can take several days or weeks to form, cast formation in our model was rapid (by 18 h) after inhalation exposure. This timing of cast formation in the SM model resembles most closely that seen after burns and smoke inhalation (39
), extensively studied in sheep (41
), where casts were formed within 24 hours of injury (39
). Although that model is similar to ours, it differs in three distinct aspects. First, casts after burns and smoke inhalation contain not only fibrin but also eosinophils and mucus (39
). Second, casts after burns and smoke inhalation contain extensive sloughed epithelium (39
), which was much less extensive in our model, appearing as distinct voids within the epithelial surface. And third, the distribution of casts throughout the tracheobronchial tree in burns and smoke injury is from the very proximal trachea down to the level of the most distal bronchioles (47
), which was not the case with CEES inhalation, wherein tracheal casts were absent.
Intrapulmonary conducting airways spanning from immediately past the carina to the level of the terminal bronchioles at airway generation 15 (49
) were the only locations in which casts were found after SM analog exposure. Microdissection of both fixed and unfixed lungs showed this similar distribution, where immobile casts were firmly attached to the surrounding epithelium in several locations along the bronchi. The respiratory bronchioles, alveolar ducts, and alveoli were free of casts, as was the trachea. Although the aerosol particle size (mass median diameter, 0.6–0.7 μm) would predict strong deposition even to the level of the alveoli, there was remarkably little evidence of injury to distal lungs noted by light microscopy, with no evidence of fibrin deposition within the lung parenchyma. By contrast, fibrin deposition was noted via IHC methods within the peribronchovascular space of airways containing plastic casts. Patchy epithelial sloughing was also observed via confocal microscopy, mainly within the mainstem bronchi and terminal bronchioles, but also along the distal trachea and respiratory bronchioles where neither casts nor fibrin deposition was seen. Although the exact mechanism for this epithelial sloughing has not yet been elucidated, we demonstrated that CEES inhalation injury is at least in part dependent on reactive oxygen and/or nitrogen species (50
). In addition, direct damage to the epithelial cytoskeleton may occur shortly after contact with the inhaled CEES compound (our unpublished data), which may not be visible via light microscopy. Whereas sloughed epithelium was noted throughout the entire airway at 18 hours, fibrin cast formation and peribronchial fibrin deposition were not. Therefore, loss of epithelial integrity is likely not the sole cause of cast formation, albeit it may be a contributing factor. Any degree of damage to the epithelium, gross or microscopic, will expose the underlying submucosal and bronchial blood vessels, potentially facilitating entry of CEES or its downstream reactive species into the local circulation, thereby facilitating injury and increased vascular permeability. Indeed, when we employed monastral blue labeling to survey for highly permeable injured vessels, we successfully localized vascular injury to the entire bronchial plexus. Interestingly, the distribution of the bronchial circulation corresponded to the distribution of cast formation and peribronchovascular fibrin deposition (noted via IHC) along the tracheobronchial tree. Together, these findings suggest that airway epithelial injury may extend to involve the underlying bronchial circulation, leading to extravasation of plasma contents such as fibrin, and activation of the clotting cascade within the airways. Although adaptive benefits of increased vascular permeability might include recruitment of inflammatory mediators and coagulation factors designed to help in repair, we believe that the increased vascular permeability occurring after SM analog inhalation is excessive, potentially contributing to deleterious acute and chronic respiratory effects seen after exposure. Therefore, we next focused on the effects of this increased bronchial vascular permeability, particularly as it relates to leakage of fibrin and other plasma-derived proteins into the airways before cast formation.
The significant increase in plasma-derived proteins within the BALF of rats exposed to the SM analog CEES indicated a vascular injury occurring early after exposure. A concentration- and time-related increase was seen between groups in BALF fibrin, total protein, and high molecular weight IgM. Although significant increases were noted, we suspected that the actual levels of these components in CEES-exposed BALF were likely underestimated, as casts were not included in the BALF analyzed. The increased levels of these proteins detected relative to the diluent-exposed group indicated a substantial vascular alteration allowing the leakage of these components out of the intravascular compartment. Evans blue dye injection in vivo
allowed us to confirm the vascular leakage by noting (via microdissection) blue-labeled albumin extravasation into the peribronchovascular region from distal trachea to terminal bronchiole (airway generation 17), corresponding to the bronchial circulation. Within these regions, histological examination revealed edema formation and immunohistochemistry revealed fibrin deposition. By 72 hours after exposure, substantial spindle cells (potentially fibroblast or myofibroblast) were noted within the peribronchovascular regions, where a large amount of fibrin was noted earlier, at 18 hours. Both mature and immature collagen deposition was seen in these areas (51
), particularly within the subepithelial interstitium of the submucosa, as well as within the surrounding regions. The pattern of collagen deposition seen 72 hours after CEES injury, together with the fibrinous obstructive cast formation with spindle cell invasion, resembles that seen in the early stages of bronchiolitis obliterans (52
). Although bronchiolitis obliterans is a known late complication after SM injury (1
), its pathogenesis remains unclear. We believe that our findings of acute epithelial, submucosal, peribronchial, and bronchial vascular injury with subsequent obstructive cast formation and collagen deposition within the intrapulmonary conducting airways may play an integral part in the development of bronchiolitis obliterans after SM inhalation. Further studies focusing on chronic lung injury model after SM exposure will be necessary to understand the evolution of this process.
The extensive involvement of the bronchial circulation in cast formation after inhalation injury has been seen and studied extensively in the sheep burn and smoke inhalation model (38
). Although SM injury differs in several respects, the similarities of early bronchial circulation involvement in cast formation seen in both models lead us to speculate that some of the underlying mechanisms could be similar. It has been proposed that three main events occurring after burn and smoke inhalation lead to early cast formation (38
). First, abundant nitric oxide production appears to lead to increased blood flow into the bronchial circulation (38
), followed by an increase in activated neutrophils that bind to the endothelium of the bronchial vessels. Thus, reactive oxygen and/or nitrogen species might be involved. Activation of adherent neutrophils could then lead to increased vessel wall permeability (38
), leading to extravasation of plasma components involved in coagulation within the airway lumen. Simultaneously, the extrinsic coagulation cascade may become activated with an increase in expression of tissue factor on pulmonary epithelial cells and alveolar macrophages (38
), allowing cast formation to be initiated. In our model, markers of inflammation appear after cast formation has begun, and only modestly. Four hours after CEES exposure, when the first signs of cast formation become evident, BALF neutrophils were scant and lung homogenate myeloperoxidase levels were virtually unchanged from control levels. At the same time, highly permeable bronchial vessels were abundant (as per monastral blue labeling), the presence of fibrin within the peribronchovascular space was pronounced, and BALF β-fibrin and protein levels were significantly elevated. By 18 hours after CEES exposure, when casts were fully formed a more pronounced inflammatory response was evident. For this reason, we believe that although inflammation was most likely a potentiator of CEES-induced airway injury, it was not the primary cause of cast formation. We have shown that oxidant injury is an important feature of CEES-induced airway injury, demonstrating strong attenuation of BALF plasma protein levels (IgM, protein) after 5% CEES exposure by administering the catalytic antioxidant compound AEOL 10150 (50
). The role of oxidant injury in cast formation after CEES inhalation will require further investigation.
Several therapeutic strategies to limit cast formation have been studied in other causes of plastic bronchitis, especially in the burn and smoke inhalation model. The main target for these therapies has been the coagulation cascade, using anticoagulant (i.e., heparin) and fibrinolytic (i.e., tissue plasminogen activator) strategies to reduce morbidity and mortality (31
). These same studies have yet to be performed after inhalation injury from SM or its surrogate, CEES. Our rat model using the SM analog CEES is a useful model for conducting such therapeutic and mechanistic studies.
In summary, we have shown that inhalation of high concentrations of the SM analog CEES in rats reliably produces airway-occlusive casts similar to those noted in the literature to cause fatal obstruction in patients exposed to SM. We demonstrated increased permeability of the bronchial circulation developing early after CEES exposure, causing leakage of plasma proteins, including abundant fibrin(ogen) into airways. The resulting airway luminal casts formed then organize within 3 days of exposure, with the appearance of early histopathologic changes suggestive of bronchiolitis obliterans. This study demonstrates and confirms the usefulness of an approach combining airway microdissection and confocal microscopy using dual vital dye staining, as originally described in ozone injury (24
), for localizing and characterizing airway injury due to toxic inhalation and for determining disease pathogenesis. In this study, we demonstrated a rat model for airway cast formation after inhalation of the SM surrogate, CEES. This model will be useful in future studies further assessing mechanisms of cast formation, potential progression to bronchiolitis obliterans, and therapeutic interventions to limit both processes (50