Inhibitor of neuronal nitric oxide synthase improves gas exchange in ventilator-induced lung injury after pneumonectomy

Postpneumonectomy pulmonary edema (PPE) is a subtype of acute lung injury (ALI) developing independently of left ventricular failure, fluid overload or infection, with a prevalence of 2.5–14.3% and a mortality rate of 50–100% [1, 2]. By inducing local release of cytokines, the lung injury is not only limited to the airways and the pulmonary vessels, but may progress to circulatory shock and multiple organ dysfunction syndrome (MODS) [3]. Recently, we noticed that pneumonectomy followed by doubling the tidal volume at zero end-expiratory pressure induces ventilator-induced lung injury (VILI) in sheep, which is characterized by derangement of gas exchange, and increments in extravascular lung water and pulmonary vascular permeability [4].

Nitric oxide (NO) generated from L-arginine by calcium-dependent endothelial NO synthase (eNOS) plays an important role in the homeostasis of circulation by modulating vascular tone [5]. Studies performed predominantly on small animals have indicated a role for NO synthesis in the pathogenesis of VILI [5–7]. Recently, investigators observed that eNOS is up-regulated after pneumonectomy in rats [8]. In rabbits, Stromberg and co-workers noticed that mechanical stretch of lung tissue might modulate the NO metabolism [9] and more recent reports revealed that both eNOS and iNOS are up-regulated in VILI in rats and mice [6, 7]. Indirect effects of NO include the formation of reactive nitrogen species that damage cells and tissues [10]. Translocation of bacteria through overstretched alveolar epithelium may represent an alternative way of activating inducible NO synthase (iNOS) during excessive ventilation, thereby enhancing the generation of NO [11]. The cytotoxic effect of NO, most likely, increases after combining with highly reactive oxygen species to form peroxynitrite, which supposedly plays an important role in the pathogenesis of MODS [5]. In a previous study, we found that infusion of methylene blue, an unspecific inhibitor of eNOS and iNOS, did not attenuate the emergence of ovine PPE [12]. This motivated us to a search for other inhibitors of NOS that could potentially protect against VILI.

Investigators recently noticed that 7-nitroindazole (NI), an inhibitor of neuronal NOS (nNOS), attenuates ALI after smoke inhalation and burns [13] as well as after inhalation of smoke followed by instillation of live bacteria into the airways of sheep [14]. Although nNOS is abundantly distributed in airway epithelial and neuronal tissues [15], it is still unsettled whether this isoform of NOS is involved in the pathogenesis of VILI after pneumonectomy, and whether NI might antagonize this particular subtype of lung injury. Thus, the aim of the present study was to find out whether NI modulates VILI after pneumonectomy in sheep.

The study was approved by the Animal Care Committee of the University of Tromsø under the Norwegian National Animal Research Authority (FOTS id 517/Pro nr 31/07).

The present study confirms our previous findings in sheep: pneumonectomy followed by one-lung ventilation with excessive tidal volumes and zero end-expiratory pressure promotes lung injury, as characterized by increased pulmonary vascular pressure and permeability, and accumulation of extravascular lung water in concert with derangements of gas exchange [4, 12]. Continuous infusion of the inhibitor of nNOS, 7-nitroindazole (NI), from two hours after the start of injurious ventilation dampened the decrease in oxygenation and the respiratory acidosis. Apparently, NI also delays the emerging increments in extravascular lung water and pulmonary vascular permeability, although without reaching significant intergroup differences.

The absence of changes in PBVI in the early postpneumonectomy period confirmed our previous finding that ovine ventilator-induced lung injury after pneumonectomy is not a result of cardiac failure or fluid volume overload [4, 12]. This notion is also supported by the observation of no significant differences in GEDVI, which is another marker of preload, between injuriously and protectively ventilated animals [19]. We speculate that the accumulation of EVLW could be caused by a combination of increased pulmonary microvascular pressure and permeability, as evidenced by the elevations of PAP, PAOP and PVPI in the injuriously ventilated groups. A decrease in vascular capacity after volume reduction surgery in the presence of transiently increased cardiac output could, at least, partly explain the rises in PAP and PAOP. However, despite the fact that no significant intergroup differences were noticed, the latter variables tended to be lower in NI-treated sheep (Table1). These findings are consistent with previous observations from our own group as well as from investigators employing a porcine model of pneumonectomy, who found that mean PAP increased significantly immediately after the lung was removed [4, 20]. Stable PAP and PAOP without any signs of lung edema in protectively ventilated sheep confirm the findings of previous workers, who were unable to provoke postpneumonectomy pulmonary edema in dogs if the left heart filling pressures were kept within normal ranges [21].

We interpret the increase in PVPI as the result of an inflammatory response to the combination of pneumonectomy and injurious ventilation. Other workers have noticed that pneumonectomy might predispose to thrombus formation and embolization of the contralateral pulmonary artery [22]. The increased pro-coagulant activity could therefore be suspected of stimulating both the coagulation and the inflammation in the forefront of the physical stress that was induced by the injurious ventilation [23, 24]. The stimulation of inflammation is supported by the observation that LIS is higher in the injuriously ventilated group, as compared to the PROTV group (Table3). Surprisingly, neutrophil infiltration was higher in the NI-treated sheep- as the only factor differing significantly between the injuriously ventilated animals. A literature search revealed no previous studies focusing on inflammation and lung histological changes after combined pneumonectomy and injurious ventilation in sheep. Our findings contrast with those reported from ovine studies of lung injury after smoke inhalation followed by bronchial instillation of live bacteria or burns, both showing improved lung histology and reduced inflammation after NI [14, 25]. We have no good explanation of these discrepancies except for the fact that LIS showed great variations and the sample size consisted of only five sheep in each group. The assumption that coagulation and inflammation might have acted together to provoke lung injury in these animals is also consistent with recent studies demonstrating that injurious ventilation alone can lead to a biotrauma, which might increase the pulmonary microvascular permeability [26]. Worthy of comment in support of the contention that inflammation is involved, is also the observation in mice that Toll-like receptor 4, which activates the innate immune system by responding to lipopolysaccharide from Gram-negative bacteria, also initiates the innate immune response to ventilator-induced lung injury [27, 28]. However, it remains to be settled if and to what extent these observations can be of any relevance to the emergence of VILI in large animals.

The increase in EVLWI is consistent with the findings of investigators who noticed a positive correlation between the degree of lung inflation and microvascular pore radius in mechanically ventilated sheep. At high inflation pressure and volume, the restriction of solute diffusion was lost resulting in a net movement of liquid into the alveoli. The authors suggested that as the lung epithelium is progressively stretched there is an opening up of water-filled channels between the alveolar cells resulting in increased extravascular water accumulation [29]. In addition to the increased pulmonary fluid filtration, mechanical ventilation has been shown to contribute to the increased EVLWI by impeding the clearance of lung lymph in anesthetized dogs and sheep [30, 31].

In the present study, Hb concentration was significantly lower in the NI-treated – as compared to the non-treated injuriously ventilated animals (Table2). Because EVLWI did not differ significantly between the injuriously ventilated groups, it is close at mind to believe that the fluid leak prompting the increase in Hb concentration, which was actually antagonized by NI, possibly could have occurred in extrapulmonary parts of the circulation. However, the mechanism by which NI could have acted to increase the microvascular reabsorption, or lymphatic clearance from other parts of the circulation, remains mere speculation and cannot be further elucidated by this study.

Protective ventilation of the remaining lung after PE together with increased FiO₂ was sufficient to maintain normal gas exchange, as previously reported from our group [12]. Although we were unable to demonstrate significant changes in the plasma concentrations of NOx (Figure 3), we speculate that enhanced local production of NO by eNOS [8] might have contributed to vasodilatation and loss of hypoxic pulmonary vasoconstriction (HPV) in atelectatic and poorly ventilated lung areas (Table3), thereby causing a decrease in arterial oxygenation. Evidently, these non-treated animals had more extensive ventilation/perfusion (V/Q) disturbances, including PaO₂/FiO₂ ratio reaching ARDS criteria, increased venous admixture, and higher alveolar dead-space, as indicated by the rise in PaCO₂. Albeit not significantly different, there was a trend towards lower C_QS and histologically more atelectases in the INJV group. In contrast, NI-treated animals displayed improvements of the deranged gas exchange (Figure 2, Table2) and less severe atelectasis. We believe that the gas exchange improved as a result of more favorable V/Q distribution due to the combined effects of reinforcement of HPV, less atelectasis and slightly increased C_QS after administration of NI, although none of the latter changes were significant. However, whether the possible reinforcement of HPV was caused by a specific inhibitory effect on nNOS or by unspecific effects on eNOS or iNOS, cannot be settled from these experiments [25]. Similar findings (i.e. attenuation of pulmonary dysfunction) have been described by our group and others by using inhibitors of various isoforms of NOS on different ovine models of acute lung injury [25, 32–34].

Since postpneumonectomy pulmonary edema occurred only in sheep subjected to injurious ventilation and not in protectively ventilated animals, we assume that ventilation with excessive tidal volumes and zero end-expiratory pressure might have played a pivotal role for this complication to develop. Consistently, we also found the most severe interstitial edema together with neutrophil infiltration and hyaline membrane formation in the injuriously ventilated groups, as confirmed by LIS (Table3, Figure 4). Our findings agree with those of clinical investigators, who described increased pulmonary microvascular permeability, loss of endothelial integrity and alveolar edema after lung resection, as well as neutrophil activation after esophagectomy and pneumonectomy [35, 36]. In contrast, investigators studying ALI in sheep after smoke inhalation in combination with airway instillation of bacteria - or with third degree burns, noticed that inhibition of nNOS both reduced the airway obstruction and improved the ventilation mechanics and the gas exchange [13, 14, 32]. Recently, the latter investigators noticed that ovine sepsis is associated with early and transient rises in the expression of eNOS and iNOS, while expression of nNOS remains unchanged [37]. Whether corresponding changes occur in response to excessive one-lung ventilation after pneumonectomy has not been settled.

The present study has several limitations: we provide no evidence of increased production of NO (Figure 3), and due to technical reasons, we were not able to determine the expressions of nNOS, eNOS and iNOS, or their proteins in lung tissue. However, higher vascular tones both in the systemic - and the pulmonary circulations, and less venous admixture in NI-treated sheep (Table1, Figure 2), suggest that at least, a partial inhibition of NOS has taken place, most likely of eNOS [8]. We also admit the lack of a group demonstrating the effects of NI alone and a lack of dose–response data, butother investigators registered no changes in hemodynamics or gas exchange that could be attributed to the use of NI per se [32]. These investigators also found that a dose of NI 1 mg/kg/h was sufficient to reduce NOx plasma levels and to preserve HPV, whereas no further increase in HPV was obtained with higher doses [25]. These investigators started administration of NI one hour after the commencement of the injurious stimulation on awake sheep over 24–48 hrs [13, 14, 25]. In our study, NI was administered from two hours after the start of injurious ventilation, which lasted only 8 hrs after the pneumonectomy. It is therefore likely that the significant effect on gas exchange and the tendency towards a significantly increased difference in EVLWI between the injuriously ventilated groups had continued beyond 8 hrs. Investigators studying the effect of NI on ovine lung injury after smoke inhalation and burn, reported the first sign of improved oxygenation between 6 and 12 hours after the injury, as assessed by a decreased intrapulmonary shunt [32]. In contrast to our first study employing the same animal model, some of the present data displayed a greater variability, which is caused, at least in part, by the fact that muscle relaxants were not used [4]. We also notice as a shortage the sample sizes of only five animals in each group examined for LIS.

Pneumonectomy followed by one-lung ventilation using high tidal volumes and zero end-expiratory pressure induced a lung injury in sheep, which is characterized by pulmonary edema and derangement of gas exchange. Intravenous infusion of the inhibitor of nNOS, 7-nitroindazole, started two hours after the pneumonectomy and the start of injurious ventilation, improved gas exchange but did not prevent lung injury with pulmonary edema in these experiments over eight hours. Further experiments of longer duration seem to be necessary to find out whether 7-nitroindazole alleviates this subtype of ventilator-induced lung injury, as experienced in other ovine models of acute lung injury.