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Acid aspiration is a major cause of acute lung injury. However, the mechanisms that underlie this spatial expansion of the injury remain undefined. In current animal models of acid injury, intratracheal acid instillation replicates the lung injury. However intratracheal instillation causes a global effect, precluding studies of how the injury spreads. Here, we report an airway catheter-based method for localized acid delivery in the isolated blood-perfused rat lung. We co-instilled hydrochloric acid with evans blue through the catheter into one lung and determined blood-free extravascular lung water in tissue samples from regions that either received, or did not receive the instilled acid. Tissue samples from the noncatheterized contralateral lung were used as controls. Lung water increased both in the regions that received acid, as well as in adjacent regions that did not. Pretreating the lung with vascular infusions of the gap junctional blocker, glycerrhetinic acid, blunted the acid-induced lung water increase at the adjacent regions. These findings indicate that endothelial gap junction communication causes spread of lung injury from regions that were directly acid injured, to adjacent sites that did not directly receive acid. Our new method for establishing localized acid injury provides evidence for a novel role for vascular gap junctions in the spatial expansion of acid injury. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.
Acute lung injury (ALI), which is a cause of high mortality and morbidity, associates with predisposing conditions such as sepsis, gastric acid aspiration, and trauma. As pathogenic mechanisms underlying ALI continue to be inadequately understood, there is considerable interest in developing animal models for mechanistic studies. Animal models of ALI are established by exposing lungs to pathogenic conditions either through the airway, or the vascular route. Several models are available (Matute-Bello et al., 2008; Matute-Bello et al., 2011). Examples are the pneumonitis model in which endotoxin is given intratracheally or intraperitoneally, or the acid-injury model in which concentrated hydrochloric acid (HCl) is given intratracheally.
Animal models are a good platform for addressing a well-known feature of ALI, namely, the rapidity with which edema develops across the lung. Although several mechanisms could account for the rapid onset, in a recent report, we showed that intercellular communication in lung microvessels plays a role (Parthasarathi et al., 2006). Uncaging-induced increases of Ca2+ spread through endothelial gap junctional channels, spreading proinflammatory effects to regions more extensive than that directly affected by the uncaging. Although these findings indicate that proinflammatory signaling spreads spatially in the lung microvascular bed, it is not clear whether similar mechanisms cause spatial spread of lung injury.
The difficulty with testing this hypothesis is that available ALI models induce lung injury in a global manner, precluding quantification of spatial spread. Here, we developed a novel lung injury model in which our objective was to deliver the injurious agent to a localized region of the lung parenchyma. As the lung parenchyma is eminently accessible to focal sampling, our plan was to cause focal lung injury, then sample lung tissue from the directly injured region as well as from adjoining regions that did not receive the injury agent. To this end, we delivered concentrated HCl through a catheter wedged in the left lower lobe of the rat lung. Our findings indicate that this approach provides a suitable model for inducing focal lung injury and that the injury spreads to the adjoining parenchyma by gap junctional mechanisms.
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Mechanisms underlying the spatial expansion of lung injury remain unclear. Here, we developed a novel, catheter-based method for localized acid-induced lung injury. Our study indicates that following focal acid instillation in a lung lobe, lung injury occurs not only directly in the acid instilled region but also in adjacent regions that did not receive the acid.
To establish the region that made direct contact with the instilled acid, we co-instilled the fluorescent marker, EB. The dye prominently demarcated the surface boundaries of the acid-treated region. Reports indicate that arterial oxygen content decreases and protein leakage increases 1-hr post-acid instillation (Folkesson and Matthay, 1997; Yamada et al., 2000; Matt et al., 2009) and endothelial permeability increases in the acid treated regions (Modelska et al., 1999). Consistent with these reports, our findings show that lung water increased at the site of acid instillation.
The novel finding was that in addition to lung injury at the focal instillation site, lung water increased in adjacent regions that did not directly receive acid. Both epithelial and endothelial mechanisms may contribute to acid injury. The absence of the markers, EB and FDx, in the adjacent regions clearly suggests that these regions did not receive any acid and hence, not directly injured by the acid. The rapid buffering of acid in airways further limits the possibility that the instilled acid spread far from the site of instillation and induced direct injury in the adjacent areas. Inhibiting endothelial gap junctional communication with intravascular GA blunted the increases in lung water at the adjacent sites. This blunting was not the result of intravascular GA leaking into airspaces before acid instillation and blocking epithelial gap junctions, as the isolated lung preparations were maintained at pressures that do not allow small solutes to cross the microvascular barrier (Parthasarathi et al., 2002). However, post-instillation, GA may have leaked into air spaces with the edema fluid. However, GA instilled into airways with acid did not inhibit the acid-induced responses at any measurement site. Hence, we interpret that inhibition of epithelial gap junctional communication by GA leaking with the edema fluid did not contribute to the reduction in lung water increases at the adjacent sites. This suggests that the acid-induced responses were independent of epithelial mechanisms. Thus, endothelial mechanisms may be predominant in increasing lung water at the acid-free regions.
At sites of direct acid injury proinflammatory second messengers might be generated in endothelial cells as a result of epithelial-endothelial crosstalk, as suggested previously for tumor necrosis factor (TNF)-treated lungs (Kuebler et al., 2000). Endothelial gap junctions might have mediated the communication of the proinflammatory mediators from the acid-treated site to adjacent uninjured sites, as previously reported (Parthasarathi et al., 2006). Thus, lung water increase at these uninjured sites might have occurred as a consequence of this communication. Thus, we interpret that interendothelial communication mediates the propagation of lung injury. While cytokines released into the circulation during injury are reported to expand injury (Tutor et al., 1994), the absence of lung water increases in the contralateral lung suggests that this mechanism was not relevant here.
To inhibit endothelial gap junctional communication, we pretreated vessels with GA. Although it is well known that GA blocks intercellular gap junctional communication (Braet et al., 2003; Earley et al., 2004; Yu et al., 2010), nonspecific effects need to be considered. GA might have induced anti-inflammatory effects (Feinstein and Schleimer, 1999; Kao et al., 2010). However, this appears to be unlikely here since at the site of direct acid injury, increases in lung water were similar for both GA-treated and -untreated lungs. Moreover, the anti-inflammatory effect of 18-alpha-GA isomer used here is small compared with that of the 18-beta-GA isomer (Antolini et al., 1992; Kroes et al., 1997), for which the anti-inflammatory effect requires hours of exposure (Kao et al., 2010). Here, GA exposure was for 10 min, which is very short for a substantial anti-inflammatory. Thus, the present lung water effect by GA was due to inhibition of endothelial gap junctional communication.
These observations extend our previous findings that interendothelial gap junctional communication is the primary mechanism for the spread of the proinflammatory responses in microvessels (Parthasarathi et al., 2006). While second messengers involved in this study remain unknown, it is possible that cytosolic Ca2+ increases mediate the spatial spread of acid injury since endothelial Ca2+ mediates permeability changes in response to thrombin, stretch, TNF, and pulmonary hypertension (Ichimura et al., 2005; Tiruppathi et al., 2006; Townsley et al., 2006; Kuebler et al., 2010). Moreover, we previously reported that alveolar TNF instillation increases vascular endothelial Ca2+ (Kuebler et al., 2000). It is possible that airway acid instillation initiates endothelial Ca2+-dependent spatial expansion of acid injury. Further studies are required to better understand the role of Ca2+ in acid injury.
In this study, we use focal instillation of acid to establish the role of endothelial gap junctions in acid injury. Other injury models such as intratracheal instillation of acid may initiate a more global injury. Even under these conditions, inhibiting gap junctional communication might protect the healthy uninjured parenchyma not directly subjected to acid contact. However, this issue remains to be resolved.
In conclusion, using a novel method of focal acid instillation, we show that focal acid injury induces lung water increases in regions within the same lung lobe but spatially distant from the site of acid instillation. Endothelial gap junctional communication mediates this spatial extension of injury. Inhibition of gap junctional communication may be therapeutic in the treatment of ALI.