Non-lobar atelectasis generates inflammation and structural alveolar injury in the surrounding healthy tissue during mechanical ventilation

Acute respiratory distress syndrome (ARDS) is characterized by prominent heterogeneous distribution of lung densities, and the regional transalveolar pressures may present marked dispersion [1],[2]. At the microscale, when an alveolus collapses the traction forces exerted on its walls by adjacent expanded units increase and concentrate. These forces may promote its re-expansion at the expense of potentially injurious stresses at the interface between the collapsed and the expanded units [1]-[3]. These inhomogeneities are also known as pressure multipliers or stress risers [1],[2],[4],[5]. This conceptual framework was described by Mead and coworkers in 1970 and is essentially related to alveolar interdependence phenomena [3],[6].

It is well-known that mechanical ventilation in itself can harm the lung, inducing or aggravating ARDS. Much debate remains, however, over pivotal concepts regarding the pathophysiology of ventilator-induced lung injury (VILI) [7],[8], especially about the precise contribution, kinetics, and primary role of regional putative VILI mechanisms [1],[2],[9],[10]. Hypothesized mechanisms of VILI include low-volume injury and over-distension of the lungs. Low-volume injury encompasses local concentration of stresses in the vicinity of collapsed regions and also cyclical recruitment of airways and alveoli [3],[10]-[14]. Over-distension injury results from increased regional lung volume and excessive deformation of epithelial and endothelial cells, as well as of the extracellular matrix, leading to a pro-inflammatory response. In some studies, low-volume injury was indicated to predominate as mechanism of damage [4],[5],[10]-[14], whereas in others including laboratory [6],[10],[15] and clinical studies [7],[8],[16],[17] over-distension was considered the predominant VILI mechanism.

In the heterogeneously expanding lung, the initial triggering mechanism of local injury and inflammation may occur predominantly in regions likely to undergo either concentration of stress or cyclic recruitment. However, there are scarce data on in vivo topographical association between regional alveolar aeration and inflammatory changes. Recently, Borges et al., using a two-hit injury ARDS animal model observed that inflammation was more pronounced within the normally and poorly aerated regions [18]. These findings challenge the classical paradigm of VILI, which claims that it occurs mainly in either over-distended or collapsed lung regions. Notwithstanding, theoretical and physiological models still suggest that atelectasis may be a decisive early event [19], acting as a key stress raiser in its surrounding in the very beginning of the inflammatory process accompanying VILI [6],[20].

We developed a rat model to test the hypothesis that local non-lobar atelectasis can act as a stress concentrator, contributing to inflammation and structural alveolar injury in the surrounding healthy lung tissue during mechanical ventilation.

Thirty-five male Wistar rats (250 to 340 g) were taken from nursery of the Instituto de Biofísica Carlos Chagas Filho - Rio de Janeiro/Brazil. All animals received care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guiding Principles in the Care and Use of Animals approved by the Council of the American Physiological Society, USA. The Ethics Committee on the Use of Animals - Health Sciences Centre from the Federal University of Rio de Janeiro approved the study (IBCCF-188-05/16).

Both ventilatory strategies presented hemodynamic stability and all animals survived until the end of the experiment. Baseline gas exchange is show in Table 1. Respiratory system mechanics and hemodynamic data over the protocol are shown in Table 2.

We choose the ventilator-group settings to evaluate the injury modulation trough a clearly injurious modality (V_T20) and a less injurious V_T setting (V_T10), but the V_T10 group resulted in a mean airway pressure 25% higher and in a respiratory system elastance 33% higher than the V_T20 group. Also, the stress index was >1.2 in the V_T10 group.

Atelectasis was successfully induced in the basal region of the lung of almost all animals (26/29 animals). The microCT images of the animals revealed that the volume of the atelectasis was 0.12 and 0.21 cm³, respectively (Figure 3). The microCT images demonstrated that there was not any other lung collapse at the start of the protocol.

In the present study we used a novel model of non-lobar atelectasis through non-selective bronchial blocking in rats, and investigated its effects on the atelectatic area as well as on the surrounding healthy lung tissue during injurious mechanical ventilation. The atelectasis model was reliable and greater histological evidence of hyperinflation and inflammation was observed in the PeriAT region.

Most of the experimental studies on the relationships between atelectasis, mechanical ventilation and VILI have the starting point of surfactant depletion and/or some type of acute lung-insult [25]. For instance, surfactant depletion is usually produced by saline lavage and results in decrease in lung compliance and hypoxemia [26]. When saline lavage is followed by mechanical ventilation with high volumes and low PEEP, a type of lung injury results that is very similar to ARDS in humans [25]. These models invariably contain, from the very beginning, unstable airspaces and a consequent gravitational gradient of collapse. In contrast, our experimental model was designated in such a way that the starting point was, as much as possible, only the primary interaction between collapsed and aerated regions, that is, before any surfactant dysfunction and/or another potential VILI mechanism could become involved. Accordingly, our model initially presented lung parenchyma homogeneously aerated with an isolated peripheral inhomogeneity. In this way we can focus on the true interaction between collapse and healthy alveoli during the mechanical ventilation.

VILI has been thought to predominate in the collapse-dependent regions and/or in the over-distended non-dependent regions. Tsuchida et al. demonstrated that in a model of alveolar collapse and surfactant depletion the atelectasis regions per se were protected from alveolar damage, whereas alveoli from the aerated regions were more affected [15]. Very recently, Cereda et al. [27] used magnetic resonance imaging to provide in vivo imaging evidence of airspace expansion in a surfactant-depleted model. Their data suggest that atelectasis could contribute to VILI by reciprocal increases in airspace size and that lung injury could be minimized through alveolar recruitment and adequate PEEP. This suggestion is supported by morphometric [15] and clinical data [28] and contrasts with previous reports suggesting an increase of VILI in the atelectasis during MV [11],[12]. Adding another point of view for the pressing question on the localization of the regional onset of lung inflammation, other recent findings suggest that the interface between the collapsed and aerated tissue may play an important role in lung injury [5].

Our findings, together with those of other investigators [29], suggest that the increased susceptibility to VILI is related to small-length-scale heterogeneities of the lung parenchyma. Indeed, Rausch et al. [30] estimated, employing synchrotron-based x-ray tomographic microscopy on isolated rat lungs, that local strains developing in alveolar walls are as much as four times higher than the global ones. Their data suggest that thin regions may become overstretched, whereas regions with tissue accumulation remain unchallenged. These data fit with our own results and strongly suggest that a tidal stretch of the healthy aerated parts can play a primary role in the activation of the inflammatory signaling cascade [18],[28].

It seems likely that the collapsed region may indirectly damage the surrounding initially-healthy aerated tissue, acting as a stress riser. We found a concentration of mechanical trauma (alveolar wall disruptions and signs of over-distension) and inflammation (neutrophilic infiltration and interstitial edema) in the region surrounding the atelectasis. In their theoretical analysis, Mead et al. estimated that the alveolar pressure at the junction of the fully collapsed and expanded alveoli could be as high as 4 to 5 times the applied pressure [6]. This landmark estimation of Mead of approximately four times local amplification was recently confirmed using synchrotron-based x-ray tomographic microscopy in a preparation of rat lungs [30], discussed above. In the same direction, Rouby et al. described that in an autopsy study [31], expanded pseudocysts were concentrated around atelectatic lung regions. We found evidence of inflammation in the atelectasis region (intravascular neutrophils and tissue cytokines), however, we did not identify signs of alveolar disruptions in this area. We speculate that it could be due to vascular injury secondary to high blood flow in a systemic inflammatory scenario that could induce endothelial activation and neutrophil adhesion, explaining the inflammatory signs in this non-ventilated portion of the lung.

We did not find significant differences in cytokine concentrations between PeriAT and control regions in the two ventilatory groups. We can postulate two possible explanations for that: 1) the duration of the protocol was too short, and 2) the technique of tissue sampling may have been unable to accurately represent the two ROIs.

The current work has limitations. First, the region of atelectasis was induced exclusively in the basal region of the lung. However, the anterior or posterior location could not be selected by the methodology and setup used. Bronchoscopy-guided plugs applied in larger animals may be a suitable alternative, allowing more topographical options for the induction of atelectasis and better reproduction of clinically relevant atelectasis in healthy lungs. Second, the duration of the ventilatory protocol was 180 minutes. Maybe a longer protocol would allow studying more precisely the inflammatory response patterns and the relationships between the ROIs. Third, a non-lobar atelectasis model was used that is different from the dependent collapse characteristically observed in ARDS patients. However, it served well to assess the spatial and time effects of localized inhomogeneity, without the concomitant presence since the starting point, of other sources of airspaces instabilities. Fourth, although blockers are biocompatible and used in the clinical setting, we cannot ignore the fact that the blockers could induce some degree of airway and or parenchymal trauma. Finally, in an ex vivo lung injury model the presence of atelectasis was associated with higher transcription of stress kinases and histological evidence of lung damage in the context of protective mechanical ventilation (low tidal volume) [19]. This finding suggests that a control for future work can be a condition characterized by the presence of atelectasis without mechanical stress (for instance, using a continuous positive airway pressure mode).

In conclusion, the present findings suggest that local non-lobar atelectasis acts as a stress concentrator, generating structural alveolar injury and inflammation in the surrounding lung tissue.

Local non-lobar atelectasis acts as a stress concentrator

Local non-lobar atelectasis generates structural alveolar injury in the surrounding healthy tissue

Local non-lobar atelectasis generates inflammation in the surrounding healthy tissue