Effects Of Inhaled Nitric Oxide Combined With Different Concentration Of Oxygen In A Rat Model Of Klebsiella Pneumoniae Pneumonia

BackgroundEndogenously produced nitric oxide (NO) is an important effector molecule in the regulation of vascular tone, neurotransmission, acute and chronic inflammation, and host defense mechanisms. The widespread expression of inducible NO synthase (iNOS) following inflammation and infection has been well characterized and accepted as a vital component of the host's adaptive response to noxious stimuli and virulent pathogens. Endogenous NO appears to contribute to host defense against infectious organisms, especially intracellular pathogens including viruses, bacteria, fungi, and parasites. Endogenous NO has important effects in the setting of inflammation, possibly exhibiting both anti-inflammatory and proinflammatory effects. For example, NO may inhibit leukocyte function, but also be cytotoxic to epithelial cells, both directly and indirectly as a paracrine mediator.Nitric oxide inhalation has been demonstrated to improve oxygenation during acute respiratory distress syndrome (ARDS) by selective pulmonary vasodilation, thereby reducing ventilation/perfusion mismatching. However, despite this effect on oxygenation, mortality rates from ARDS do not seem to be reduced by NO inhalation. In addition, NO inhalation decreased leukocyte-endothelium adherence and associated acute lung injury (ALI) in animal models of ALL On the other hand, local defenses could be impaired because neutrophils are the major factor of bacterial clearance.In experimental models, inhaled NO decreased lung parenchymal damage, alveolar macrophage and neutrophil function, and the transendothelial migration of activated neutrphils during ALI. In contrast, NO and nitrite interacted with neutrophil myeloperoxidase to stimulate oxidative reactions during inflammation. Moreover, NO pretreatment potentiated ALI in an isolated rabbit lung model of oleic acid-induced ALI. These findings suggest that NO can exert detrimental as well as beneficial effects. The mechanisms involved in the variation in NO effects leading to ALI are not clear. These disparate effects could be related to differences in the timing of administration and/or in the concentration of inhaled NO or to model differences. NOadministration before the establishment of lung injury has been suggested to protect against ALI by causing a decrease in the availability of oxygen radicals, as well as by modulation of the interaction between the vascular endothelium and inflammatory cells. Recent data have indicated that reducing the concentration of NO might attenuete its potentially deleterious effects. Inhaled NO at 10-20 parts/million (ppm) attenuates lung injury and does not result in any toxic effects in human and animal models.Neutrophils infiltration in the lung, followed by the extracellular release of reactive oxygen species (ROS) and proteolytic enzymes, proinflammatory cytokine activity in the lung, and the endothelial cell expression of intercellular adhesion molecule-1 have been considered as primary events in the pathogenesis of ALI. However, the molecular and cellular mechanisms underlying the effects of inhaled NO on the pathogenesis of ALI in vivo still remain to be clarified.One of the mechanisms by which NO may modulate lung inflammation is through its interaction with the transcription factor nuclear factor (NF)- k B. NF- k. B is a multiprotein complex that regulates a variety of diverse genes, including interleukin (IL)-l, -2, -6, and -8, tumor necrosis factor (TNF)-a, various adhesion molecules, acute-phase proteins, proteinases, and iNOS, which play important roles in the initiation and progression of lipopolysaccharide-induced ALI. Recent evidence indicates that intraperitoneal administration of endotoxin in rats leads to the activation of NF- k B in lung tissue, which is associated with lung neutrophilia, increased epithelial permeability, and lipid peroxidation.Few studies have been reported in the effects of NO inhalation on inflammatory response of lung in bacterial pneumonia. These effects are of particular importance because bacterial pneumonia is a major cause of ARDS and nosocomial bacterial pneumonias frequently occur during the course of this syndrome. Presently, the effects of NO inhalation on bacterial clearance and inflammatory response of lung in the setting of pneumonia caused by gram-negative bacteria has not been clearly defined. In this study, we sought to assess the effects of NO inhalation associated with air or various FiO2 (a more relevant situation regarding patients with ARDS) on pathophysiology, bacterial clearance, inflammatory response, metabolisms of surfactant phospholipids and pulmonary NOS activity in a rat model of K. pneumoniae pneumonia.ObjectivesTo investigate the effects of inhaled NO combined with different concentration of oxygen in a rat model of K. pneumoniae pneumonia1. on pulmonary bacterial clearance.2. on lung inflammation and their mechanisms.3. on pulmonary sunfactant phospholipids content and function4. on endogenous NO system.MethodsAdult healthy male SD rats (180-220 g) were used to induce bacterial pneumonia by intratracheal instillation of ESBL-producing K.pneumoniae from patients in our study. After instillation, the rats were randomly exposed to air, inhaled NO 20 ppm, 40% oxygen, 40% oxygen plus inhaled NO, pure oxygen or high oxygen plus inhaled NO groups during 24 hours. In addition, sham animals were randomly assigned to air or inhaled NO groups. After exposure, We measured arterial blood gas, blood white cell count, plasma and bronchoalveolar lavage fluid (BALF) levels of NO metabolites, leukocyte count, protein concentration and phospholipids content in BALF, and pulmonary myeloperoxidase (MPO), NOS and NF- k B activity, intercellular adhesion molecule (ICAM-1) and tumor necrosis factor (TNF-a) levels. We also measured pulmonary TNF-a, ICAM-1, endothelial NOS (eNOS) and iNOS mRNA expression by RT-PCR method. Bacteriologic culture of blood, and lung was performed. Meanwhile, pathologic changes were examined.Results1. Physiological and pathological indicatorsK. pneumoniae-infected rats appeared tachypneic and wheezy, had ruffled fur, and had minimal interaction with their environment. Gas exchange was impaired after induction of pneumonia as PaO2 and SaO2 was decreased in pneumonia vs. sham rats (P < 0.01), but pH, HCO3", PaCO2 and white blood cell counts were not significantly different between sham and pneumonia rats. Pneumonia rats exposed to inhaled NO plus high oxygen had the highest PaCO2 and HCO3" in pneumonia groups exposed to various gases. Inhaled NO, higher concentration oxygen or both combination improved oxygenation to various extent compared with air exposure.Histopathological examination of lungs under light microscopy showed that themain pneumonia feature was an infiltration of leukocyte in interstitial and alveolar spaces. Extensive peribronchial inflammatory exudation also expanded through adjacent alveoli. Inhaled NO in normal rats produced mild inflammatory response, while inhaled NO in pneumonia rats markedly reduced leukocyte infiltration, but high oxygen plus inhaled NO tended to increase leukocyte infiltration. There was not significant edema in the lung sections of pneumonia rats, and no significant difference in wet/dry lung weight ratio was observed between pneumonia rats and sham rats (P > 0.05), but high oxygen significantly increased water content of the lungs in pneumonia rats.2. Bacteriologic culture of blood, BALF and lungsHemoculture of sham rats was negative, but positive rates in pneumonia rats exposed to air and to inhaled NO were 44.4% (4/9) and 55.5% (5/9) respectively, but positive rate of pneumonia rats in 40%O2, inhaled NO plus 40%O2, high O2 and inhaled NO plus high O2 groups were 22.2% (2/9), 20%(2/10), 12.5% (1/9) and 0 (0/8), respectively. Cultures of BALF and Lung homogenates in pneumonia rats showed that bacterial counts in rats exposed to air, 40% oxygen and pure oxygen were significantly higher than those in rats exposed to corresponding plus inhaled NO, respectively (P< 0.01).3. Effect of NO on K. pneumoniae cultures in vitroExposure of serial dilutions of K. pneumoniae cultures in vitro to 20 ppm of NO vs. air for 18 hrs indicated a significant inhibitory effect of NO on culmulative growth over 18 hrs at the higher dilutions (104 and 105cfu/ml). Serial assessment of bacterial counts in cultures exposed to 20 ppm NO vs. air for 24 hrs showed simiar growth during the first 12 hrs of the 24 hrs exposure period. In contrast, during the final 12 hrs of the 24 hrs period of exposure to NO, an antibacterial effect of NO became apperent(P<0.01).4. Leukocyte recruitmentAlveolar macrophages (AM) were the predominant resident leukocytes in alveolar spaces of noninfected animals, and only scarce polymorphonuclear leukocyte (PMN) could be recovered from that site. Infection triggered neutrophils recruitment into alveoli of rats, resulting in high leukocyte counts in pneumonia rats. Inhaled NO effectively prevented neutrophils recruitment in comparison with air (P < 0.01), in contrast, a higher PMN count in BALF was observed in high oxygen plus inhaled NO group than that in pure oxygen group.MPO activity in two groups of sham rats was similar. Pulmonary MPO activity in pneumonia rats was significantly higher than that in sham rats (P < 0.01), and inhaled NO exposure markedly reduced pulmonary MPO activity compared with exposure to air in pneumonia rats (P < 0.01). High oxygen plus inhaled NO increased MPO activity in pneumonia rats in comparison with high oxygen (P < 0.01). 40% oxygen plus inhaled NO mildly reduced pulmonary MPO activity compared with 40% oxygen (P > 0.05).5. Protein levels and analysis of phospholipids in BALFProtein levels in BALF were significantly increased in pneumonia rats compared with sham rats (P < 0.01), and high oxygen further increased protein levels in pneumonia rats. Protein levels in pneumonia groups exposed to plus inhaled NO were significantly lower than those in pneumonia groups exposed to corresponding non-inhaled NO, respectively (P < 0.05 or P < 0.01). TPL and DSPC content in pneumonia rats were markedly lower than that in sham rats, and oxygen exposure significantly deceased DSPC to TPL ratio. K. pneumoniae-induced pneumonia impaired surfactant function, and Inhaled NO had no significant influence on TPL and DSPC content and their function in BALF.6. Effects of K. pneumoniae and iNO on TNF-a and ICAM-1Pulmonary levels of TNF-a after inhaled nitric oxide in normal rats were higher than those of rats exposed to air. Bacterial-induced pneumonia significantly increased pulmonary levels of TNF-a compared with normal rats, all of inhaled NO and 40% oxygen and 40% oxygen plus inhaled NO markedly reduced pulmonary levels of TNF-a in pneumonia rats. However, high oxygen plus inhaled NO increased pulmonary levels of TNF-a compared with high oxygen. The change of pulmonary levels of TNF-a nearly paralleled that of MPO activity in lung homogenates. But there was no difference in TNF-a mRNA expression between groups. ELISA results indicated that pulmonary ICAM-1 levels in normal rats were low, and bacterial-induced pneumonia significantly increased pulmonary ICAM-1 levels. Inhaled NO associated with various concentration of oxygen significantly decreased pulmonary ICAM-1 levels compared with corresponding concentration of oxygen without NO inhalation, respectively. Moreover, the change of pulmonary ICAM-1 mRNA expression was similar to that of pulmonary ICAM-1 levels, although there was no difference in ICAM-1 mRNA expression between groups.7. Effects of K. pneumoniae, inhaled NO and oxygen on endogenous NO systemExcept that there was a marked decease in pulmonary total NOS activity in pneumonia rats exposed to air (P < 0.01), no significant difference was observed between other seven groups. Relative to sham rats, induction of pneumonia by K. pneumoniae increased pulmonary iNOS activity (P < 0.01) with a concomitant decrease in cNOS activity (P < 0.01). Inhaled NO, 40% oxygen and inhaled NO plus 40% oxygen alleviated inhibition of cNOS activity by K. pneumoniae-induction (P < 0.01), while there was no significant influence on pulmonary iNOS activity. High oxygen, especially high oxygen plus inhaled NO, significantly inhibited increased iNOS activity, and recovered inhibited cNOS activity in pneumonia rats. However, K. pneumoniae and inhaled various gases had no influence on pulmonary eNOS and iNOS mRN A expression.Levels of MetHb in blood in inhaled NO rats were significantly higher than those in non-inhaled NO rats (P < 0.01). Plasma levels of nitrite/nitrate in pneumonia rats were markedly higher than those in sham rats, and inhaled NO seem not to increase the levels of nitrite/nitrate in plasma. That inhaled NO significantly increased BALF levels of nitrite/nitrate in normal animals (P < 0.01) was similar to that occurred in K. pneumoniae pneumonia rats, but inhaled NO mildly increased BALF levels of nitrite/nitrate compared with non-inhaled NO in pneumonia rats (P > 0.05). 8. inhaled NO and NF-kB binding activityPulmonary NF-kB activity significantly deceased after inhalation of nitric oxide in sham rats in comparation with control group (P < 0.01). Pulmonary NF-kB activity was significantly higher in pneumonia rats than that in normal control rats (P < 0.01).Pulmonary NF-kB activity was significantly lower in both inhaled nitric oxide plus air and plus 40% oxygen groups than that in both air and 40% oxygen groups, respetively, While there was no significantly difference in Pulmonary NF-kB activity between high oxygen and high oxygen plus inhaled NO groups.Conclusions1. Inhaled NO 20 ppm for 24 hours induced mild lung inflammation in healthy rat lungs.2. Inhaled NO enhanced clearance of K. pneumoniae, and inhalation of higher concentration oxygen might reduce development of bacteriemia.3. Combination of inhaled NO with lower concentration of oxygen decreased pulmonary leukocyte infiltration, and attenuated lung injury, while combination...