Treatment with inhaled nitric oxide has been studied as an adjuvant therapy for a variety of pulmonary conditions. Because inhaled nitric oxide (NO) acts as a potent selective pulmonary vasodilator (1) that produces pulmonary vasodilatation in well-ventilated lung regions, it can potentially improve matching ventilation and perfusion (2, 3). However, up to 60% of patients with septic acute respiratory distress syndrome (ARDS) do not respond, or respond only minimally, to inhaled NO with improved oxygenation (4). Recent reports suggest that increased NO synthesis in the septic lung causes a poor responsiveness to inhaled NO (5). Attenuation of hypoxic pulmonary vasoconstriction may be another determinant of the non-response phenomenon to inhaled NO in the septic lung (6). Some studies have demonstrated that NO-dependent guanylated cyclase activity plays an important role in attenuating hypoxic vasoconstriction in the rabbit lung (7). Fischer et al. (8) reported that nitric oxide synthase inhibition restored hypoxic pulmonary vasoconstriction in sepsis. These reports suggest that improvement of oxygenation with inhaled NO may be augmented by blockade of endogenous nitric oxide synthase (NOS) in the setting of increased NO synthesis in the lung, such as pneumonia (9) or sepsis (10).

Prior studies on the effects of combined treatment, with blockade of NOS and NO inhalation, have been reported on both neonatal and adult models of experimental sepsis and endotoxemia (10-12), where recovery of the systemic blood pressure by blockade of NOS and simultaneous attenuation of pulmonary hypertension by NO inhalation were targeted. The findings show partially beneficial effects and suggest that this approach deserves further evaluation (11, 12). However, it is not known whether NOS blockade, in hemodynamically stable lung injury, is associated with improved oxygenation with NO inhalation. There are concerns that this approach might aggravate pulmonary hypertension, induced by NOS blockade, and compromise further oxygenation, especially in newborns.

We developed a newborn piglet model of Gram-negative bacterial pneumonia and sepsis by endotracheal instillation of Escherichia coli previously (13), which is thought to be appropriate to study these concerns. Because confined inflammation occurs, in the early phase of pneumonia, only in the lung, animals using this model are more hemodynamically stable in the early period of experiment than if the lung infection resulted from sepsis induced by intravenous infusion of bacteria. Gram-negative bacterial pneumonia with sepsis is a common and clinically important pulmonary disease associated with significant morbidity and mortality rates in humans including neonates. A recent study has reported that E. coli is responsible for one half of the early-onset blood stream infection (BSI) cases among neonates admitted to the neonatal intensive care unit (NICU) (14). BSI is a significant risk factor for the development of ventilator-associated pneumonia in neonates admitted in NICU (15). E. coli is the most common organism isolated from cultures of lung aspirates from neonates who are stillborn or die in the first 72 hr of life in developing countries (16). Therefore this model may be clinically relevant.

We performed this study to evaluate whether the pretreatment of nitric oxide synthase inhibitor can affect the responses to inhaled NO (iNO) treatment in gas exchange and hemodynamic profiles of newborn piglets with hemodynamically stable lung injury as a result of E. coli pneumonia and sepsis.

The protocol was approved by the Research Animal Laboratory Committee of Samsung Biomedical Research Institute, Seoul, Korea, and the procedures followed were in accord with institutional guidelines. Studies were performed on 7-10 day-old newborn piglets of mixed strain (Yorkshir, conventional breed, purchased from Paju farm, Paju, Kyungki-Do, Korea). Surgical preparation of neonatal piglets was initiated by sedation with ketamine (20 mg/kg intramuscular injection) and xylazine (2 mg/kg intramuscular injection) followed by thiopental anesthesia (5 mg/kg, intravenous injection). After local injection with lidocaine (1%), a tracheostomy was performed and the piglet was paralyzed with pancuronium (0.1 mg/kg, intravenous injection.) followed by hourly intravenous injections. Sedation was maintained with hourly doses of thiopental. The paralyzed piglet was placed on a time-cycled pressure-limited infant ventilator (Sechrist Infant Ventilator, Model IV-100V, Sechrist Industries, Anaheim, CA, U.S.A.) to attain an arterial O₂ tension of 80-100 mm Hg and an arterial CO₂ tension of 35-45 mm Hg. The right femoral artery was cannulated for arterial blood gas sampling and systemic arterial blood pressure monitoring. The right femoral vein was cannulated into the right atrium for infusion of ice-saline to monitor cardiac output which was calculated by the thermodilution method using the CO-set (Edwards Lifesciences, Irvine, CA, U.S.A.), and for administration of fluids and medications. A 5-Fr. Swan-Ganz catheter (Baxter Health-care Corp., Irvine, CA, U.S.A.) was inserted into the right external jugular vein and advanced into the pulmonary artery using direct-pressure and pressure wave monitoring. It was used for sampling of mixed venous blood and measurement of pulmonary arterial wedge pressures. An infusion of 0.9% saline containing 1 U of heparin/mL was provided at 1-2 mL/hr through the arterial catheter and the pulmonary arterial catheter, both of which were attached to a blood pressure transducer (Hewlett-Packard Model M1276A, MA, U.S.A.). A Hewlett Packard neonatal monitoring system (Hewlett-Packard Model M1276A) continuously monitored electrocardiogram, oxygen saturation and systemic arterial and pulmonary arterial pressure. Animals were maintained supine with the head of the bed elevated 20 degrees throughout the study. Constant body temperature was maintained between 38-39℃ using a warmed operating table and servo-controlled overhead heater (Airshields, Neonatal intensive care unit, Hatboro, PA, U.S.A.).

Prior to the study, no differences were observed among five groups of animals for body weight (CON group 4.2±0.4 kg, PCON group 4.6±0.7 kg, PNO group 4.3±0.5 kg, PNA group 4.6±0.9 kg, PNANO group 4.5±0.7 kg) and total hemoglobin concentration (CON group 11.3±1.1 g/dL, PCON group 11.6±2.4 g/dL, PNO group 11.1±1.4 g/dL, PNA group 11.8±2.5 g/dL, PNANO group 12.0±4.4 g/dL). Hemoglobin remained constant in all groups throughout the study period.

We have demonstrated that L-NAME pretreatment potentiates the effect of NO inhalation, especially oxygenation in a newborn piglet model of E. coli pneumonia and sepsis. Augmented oxygenation, during iNO with pretreated L-NAME in infected animals, did not result from stabilization of circulatory instability or restoration of low blood pressure by L-NAME pre-treatment. This is because this newborn piglet model of E. coli pneumonia was not accompanied by circulatory collapse during the 6-hr study period. The study effects were associated with augmented gas exchange, complete decrease in intrapulmonary shunting and partial decrease in the relative dead space during ventilation of the infected animals as a result of combined treatment with pre-blockade of NOS and iNO, which was more pronounced when compared to infected animals treated with iNO only. In this model of pneumonia and sepsis, there was no significant changes observed in systemic and pulmonary arterial pressure during the 6-hr study period after intratracheal instillation of bacteria, however, there was a minor decrease in cardiac index, which caused a slight increase in SVRI and PVRI. INO did not affect the systemic and pulmonary hemodynamics. This is likely due to iNO not alleviating the decreased cardiac index. A decreased cardiac index might have resulted from gradual decrease in arterial pH in our study model. Further decrease in the cardiac index in the PNANO group might have resulted from aggravated systemic vasoconstriction and lactic acidosis from L-NAME pretreatment. The observed NO-related changes in oxygenation, that were seen in this piglet model with Gram-negative pneumonia and sepsis, are consistent with findings of clinical studies of acute respiratory distress syndrome, showing that inhaled NO may improve oxygenation without observed changes in pulmonary arterial pressure (19).

L-NAME pretreatment, in infected animals, was not compatible with survival and only two of 8 animals survived in that study group. This is consistent with our previous study. In that study we demonstrated that nonselective inhibition of NOS only is detrimental in the early phase of experimental neonatal sepsis (20). However, differences from this study are model used. In previous study sepsis was induced by intravenous injection of E. coli, which showed systemic hypotension (20). However in this study we used pneumonia and sepsis model without systemic hypotension, which was induced by intratracheal instillation of E. coli.

Hypoxemia during pneumonia, and other lung injury, results in large part from mismatch of perfusion and ventilation (21). Hypoxic pulmonary vasoconstriction (HPV), the normal physiological response that diverts blood flow from poorly to well-ventilated lung regions, has been shown to be impaired in patients with pneumonia, sepsis or the acute respiratory distress syndrome (22). This attenuation of HPV increases intrapulmonary shunting that results in systemic hypoxemia (8, 22, 23). Nitric oxide is known as one of the vasoactive mediators that alter pulmonary vascular tone during lung inflammation (24). Endogenous NO is produced by three different isoforms of nitric oxide synthase (NOS) that have been characterized as neuronal (NOS1), inducible (NOS2) and endothelial (NOS3) forms (25). Transcriptionally regulated NOS2-derived NO production reduces the pulmonary responsiveness to exogenous inhaled NO, alters pulmonary blood flow distribution, and impairs arterial oxygenation in endotoxin challenged mice (26).

Some possible explanations for this beneficial effect on oxygenation of L-NAME pretreatment added to NO inhalation are as follows. Previously, Fischer et al. has reported that NOS inhibition partly restores HPV in sepsis (8). In healthy animals, NOS inhibitors augment HPV (27). Very little is known, however, about the effect of NOS inhibitors on HPV in sepsis or pneumonia. Maurenbrecher et al. (6) has reported that response to inhaled NO is abolished over time and that this non-response to inhaled NO is likely to be related to the degree of V/Q mismatch. This may suggest an important role of HPV in endotoxin-induced lung injury. Recently Spohr et al. demonstrated that, in a murine model of endotoxemia, NOS2-derived NO production is critical for LPS-mediated development of impaired HPV (28).

In the pneumonia and sepsis model used in this study intrapulmonary shunting increased and arterial oxygen tension decreased progressively with time. Inhaled NO effectively decreased this V/Q mismatch, which resulted in amelioration of progressive hypoxemia. Inhaled NO added to L-NAME pretreatment, in infected animals, showed earlier and maximal decrease in intrapulmonary shunting. A previously published study by Putensen et al. (29) supports our findings. They demonstrated that in oleic acid-induced lung injury, in an adult canine model, improved ventilation-perfusion matching and gas exchange was most pronounced when NO was inhaled in the presence of systemic N-monomethyl-L-arginine (L-NMMA), a non-selective NOS inhibitor. Therefore, L-NAME pretreatment might restore HPV via inhibition of excessive production of endogenous nitric oxide, especially by NOS2, which augmented improved oxygenation with inhaled NO in our experimental model of neonatal pneumonia and sepsis.

A second plausible explanation is that L-NAME might attenuate a microvascular leak from a ventilator induced lung injury (VILI). A recent report suggested that L-NAME may attenuate ventilator-induced microvascular leaks and lipid peroxidation, and that NO may contribute to the development of VILI (30). In our experiment, all animals were ventilated with re-adjusted settings of high PIP, and high oxygen concentration right after bacterial or saline instillation. This was done to avoid significant hypoxemia that could cause additional deterioration of pulmonary hemodynamics. Therefore although the experiment duration was 6 hr, some degree of VILI, as a result of high ventilator settings, might have contributed to the lung injury especially in infected animals. L-NAME has a long plasma half-life of about 22 hr (31). In our pneumonia model, L-NAME pretreatment added to iNO, ameliorated hypercapnea and partly decreased the ratio of dead space to tidal volume. Therefore, there is a possibility that NOS blockade might blunt VILI during the study period, and potentiate the effect of inhaled NO in this model.

In summary, intratracheal E. coli instillation in newborn piglets resulted in progressive hypoxemia and hypercapnea with relatively minor changes in pulmonary and systemic hemodynamics. In this model of experimental neonatal Gram-negative bacterial pneumonia and sepsis, inhaled NO attenuated progressive hypoxemia via decrease in intrapulmonary shunting, but did not affect the increase in PVRI. Prior nonspecific NOS blockade, with L-NAME and treatment with exogenous iNO, resulted in resolution of progressive hypoxemia in this pneumonia and sepsis model and improved hypercapnea partially, although it was associated with aggravated elevation of PVRI and SVRI. Extrapolation of these findings to the clinical setting, suggest that iNO may have a limited therapeutic role in acute lung injury associated with neonatal Gram-negative bacterial pneumonia and sepsis. Endogenous NOS blockade may be a possible option for enhancement of inhaled NO, improving gas exchange in acute lung injury associated with neonatal Gram-negative bacterial pneumonia and sepsis.