SELECTIVE INDUCIBLE NITRIC OXIDE SYNTHASE INHIBITION DURING ... : Shock (original) (raw)
INTRODUCTION
The overproduction of nitric oxide (NO) has been discovered as an important factor contributing to many of the manifestations of septic shock, including hypotension (1) and vascular hyporesponsiveness to vasoconstrictor agents (2), myocardial depression, microvascular hyperpermeability, intestinal barrier dysfunction, and liver damage (Refs. 3–5 for review). The generation of large quantities of NO as a result of inducible NO synthase (iNOS) expression importantly contributes to the hypotension associated with endotoxic shock (6), and inhibition of iNOS has been shown to reverse sepsis-induced circulatory failure (7) and organ injury (8). The pathophysiological role of NO has recently been supported by a clinical study clearly documenting an association between NO overproduction, mitochondrial dysfunction, and organ failure in patients with sepsis (9). Nevertheless, various experimental studies targeting blockade of NO production provided conflicting results (3). It is generally assumed that NO produced by endothelial constitutive NOS (ecNOS) has protective effects, whereas excess NO production by iNOS contributes to the development of tissue injury. However, even iNOS-generated NO may be beneficial under some circumstances. These paradoxes are most pronounced in sepsis, where NO has a dual role, ranging from vasoregulation and cell signaling to direct cellular toxicity. Depending on the type of insult, the tissue type, the level and duration of iNOS expression, and the redox stress, NO can be either cytoprotective or cytotoxic, acting either pro- or anti-inflammatory (3–5, 10). This dual ability of NO, as well as different experimental conditions, account for these apparent divergent results. In this context, the most abundant data are derived from acute, hypodynamic, unresuscitated rodent models challenged with a large dose of endotoxin. These models are, however, criticized for their limited clinical relevance, and many emerging therapeutic approaches that have been found effective in these models failed to yield a benefit in large animal models (11). In contrast to rodent model of sepsis, much less is known about the role of iNOS-derived NO in clinically relevant large animal models. In porcine long-term hyperdynamic endotoxemia, we have recently showed that selective iNOS inhibition with 1400W significantly reduced the detrimental consequences of endotoxin on intestinal and hepatocellular energy balance (12). However, limited data from (mostly rodent) bacteremia models suggest that the consequences of NO overproduction seem to be less established compared with endotoxemic models (3). Thus, the role of iNOS in animal models that use a live bacterial challenge warrants further investigation. The current study investigated whether the protective effects of iNOS inhibition observed in our previous study (12) can be confirmed in the same experimental setup, where hyperdynamic sepsis is caused by live Pseudomonas aeruginosa. To block excess NO, the selective iNOS inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL) was used. This compound has been shown effective in preventing hypotension (but not organ dysfunction) (13) and in attenuating metabolic derangements in endotoxin-challenged rats (14).
MATERIALS AND METHODS
Animals and preparations
The experiment was performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals. The study protocol was approved by the University Animal Care Committee. Sixteen domestic pigs of either sex with a median body weight of 24 kg (interquartiles 22;24) were investigated. The surgical preparation of the animals has been previously described in detail (12, 15). The surgical anesthesia was maintained with continuous intravenous thiopental (10 mg/kg/h) and fentanyl (10 μg/kg/h) until the end of the operation. Thereafter, the continuous thiopental infusion (5 mg/kg/h) was maintained until the end of the experiment, and buprenorphine (0.3 mg) was added every 4 h and before any noxious stimuli to prevent a rise in heart rate and arterial pressure due to inadequate anesthesia. Muscle paralysis was achieved with pancuronium (4 mg/h). The pigs were mechanically ventilated (FiO2 0.4; PEEP 5 cmH2O; Servo 900C; Siemens, Brommo, Sweden) with a tidal volume of 12 mL/kg, and the respiratory rate was adjusted (14-18 bpm) to maintain arterial PCO2 between 4.7 and 6.5 kPa. During surgery, the animals received Ringer’s lactate solution (15 mL/kg/h). After surgery, the rate of Ringer’s lactate infusion was reduced to 10 mL/kg/h. A 3-Fr thermistor-tipped fiber optic catheter was inserted into the left femoral artery for continuous blood pressure recordings, blood sampling, and for thermal-dye double indicator dilution measurements. A central venous catheter for drugs and fluid infusion was inserted through the left jugular vein. A balloon-tipped thermodilution pulmonary artery catheter was placed via the right jugular vein. A midline laparotomy was performed, and precalibrated ultrasound transit time flow probes (Transonic Systems, Ithaca, NY) were placed around the portal vein, the common hepatic artery, and the superior mesenteric artery. Catheters were introduced into the portal and mesenteric veins, and an angiography catheter was placed via the right jugular vein into a hepatic vein under ultrasound guidance. A loop-ileostomy was performed, which allowed for the simultaneous insertion of a tonometry catheter (TRIP NGS catheter; Tonometrics, Helsinki, Finland) and a Doppler probe (PF 409; Perimed, Jarfalla, Sweden). The abdominal wall was then closed. In addition, a cystostomy catheter for urine collection was percutaneously placed under ultrasound guidance. A postsurgical stabilization period of 6 h was allowed before baseline measurements were obtained.
Measurements and calculations
The measurement of systemic hemodynamics included cardiac output, systemic vascular resistance, intrathoracic blood volume, and filling pressures of both ventricles. In the hepatosplanchnic region, we measured mesenteric arterial, portal venous, and hepatic arterial blood flows. Arterial and mesenteric, portal, hepatic, and mixed venous blood samples were analyzed for PO2, PCO2, and for hemoglobin oxygen saturation. Systemic oxygen delivery, systemic oxygen uptake, intestinal and hepatic oxygen delivery, and oxygen uptake were derived from the appropriate blood gases and flow measurements (12, 15). Ileal mucosal PCO2 was measured semicontinuously (time equilibration = 10 min) by automated air tonometry (Tonocap; Datex-Ohmeda, Helsinky, Finland). The ileal mucosal microcirculation was evaluated with a laser Doppler flowmetry (Periflux 4001 Master; Perimed, Stockholm, Sweden) using a probe device for the measurement of gut mucosal perfusion (PF409) (16). Each measurement represented the mean value of blood flow obtained from five randomly chosen locations, each recorded for 45 s with an optimal intensity of the backscattered light. Before each data collection, a calibration was performed using a motility standard (provided by Perimed). The probe was then advanced into the ileal lumen (10-15 cm distally). The quality of the laser Doppler signal was controlled online on a computer screen so that any motion disturbances as well as noise due to inadequate probe position could be detected before the measurement was started. Before each new area of the ileal mucosa was evaluated, the probe had been moved back or forward (at a distance of 1-2 cm). When the data for given time point had been obtained, the probe was removed from the lumen. As reported previously (12), we also determined the arterial, mesenteric, portal, and hepatic venous lactate concentrations, mesenteric and hepatic venous lactate/pyruvate ratios, and intestinal and hepatic lactate fluxes. In addition, analysis of hepatic venous ketone bodies (acetoacetate + β-hydroxybutyrate) were performed to calculate hepatic venous ketone body ratio expressed as acetoacetate/β-hydroxybutyrate (cyclic enzymatic method; Wako Chemicals, Osaka, Japan). Arterial serum levels of alanine aminotransferase (ALT, a marker for hepatocellular injury) and creatinine (a marker for renal dysfunction) were determined using a commercially available kit. Arterial nitrate + nitrite (NOx) concentrations were measured using the chemiluminescence analyzer (12, 17). Arterial 8-isoprostane (8-epi Prostaglandin F2α) levels were determined as a direct marker of lipid peroxidation using a commercial enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) (15). To correct for dilutional effects resulting from volume resuscitation, NOx, isoprostane, and ALT concentrations were normalized for plasma protein content (18).
Protocol
After recording baseline measurements, the continuous infusion of live P. aeruginosa (1 × 109 colony forming units/mL) was started until the mean pulmonary artery pressure (MPAP) reached 45 mmHg. The infusion rate was then titrated during the next 12 h, i.e., until the second data collection, to result in moderate pulmonary hypertension with an MPAP of 35 of 40 mmHg. The dose of P. aeruginosa required to achieve this target was not changed during the remainder of the study. A second set of measurements was obtained after 12 h of P. aeruginosa infusion. The pigs were then randomly divided into two groups: control animals without additional treatment except for volume resuscitation (n = 8) and animals receiving L-NIL (n = 8). This iNOS inhibitor, which has a 28-fold selectivity toward iNOS (19) without affecting baseline arterial pressure (14), was administered with a loading dose of 0.5 mg/kg over 10 min and was continued at a rate between 1 and 2 mg/kg/h to maintain mean arterial pressure at baseline levels. This dose was proven effective in reducing the renal dysfunction and injury associated with renal ischemia/reperfusion in rats (20). Moreover, the selection of the dose of L-NIL was based on a pilot dose-response experiment. Hydroxyethyl starch was administered as required to keep mean arterial pressure >60 mmHg, and 20% glucose was infused to maintain arterial blood glucose levels between 5 and 7 mmol/L. Further data acquisition was performed at 18 and 24 h after the start of P. aeruginosa infusion. When the last set of data had been obtained, the animals were killed by KCl injection under deep anesthesia.
Statistical analysis
All values shown are median and interquartile range unless otherwise stated. Differences within each group, i.e., between values before P. aeruginosa infusion and measurements during P. aeruginosa administration alone or during L-NIL infusion, respectively, were tested using a Friedman repeated measures analysis of variance on ranks and a subsequent Dunn’s test for multiple comparisons. Differences between the groups were analyzed using a Mann-Whitney rank sum test. P < 0.05 was regarded as significant.
RESULTS
There were no statistically significant differences in any measured variables between the two groups at baseline. The total number of viable P. aeruginosa (1 × 109 cfu/mL) infused in both groups was similar (control group 19 [16;21], L-NIL group 17 [16;20] mL/24 h). In the control group, infusion of live bacteria resulted in a significant gradual increase in arterial NOx levels measured as a surrogate for total body NO formation. By contrast, animals treated with L-NIL at 12 h after intervention exhibited significantly lower NOx levels in comparison with the untreated pigs (Table 1).
Nitric oxide production, systemic hemodynamic, and oxygen exchange variables
Systemic hemodynamics
Systemic and pulmonary hemodynamics and oxygen exchange parameters are summarized in Table 1. All animals developed hyperdynamic circulation with reduced systemic vascular resistance within the first 12 h of the experiment. Despite a sustained increase in cardiac output, P. aeruginosa infusion resulted in a significant gradual fall in mean arterial pressure in the control group, whereas the blood pressure in pigs that had received L-NIL was restored to baseline values. Adequate fluid resuscitation was ensured by monitoring intrathoracic blood volume, which significantly increased in both groups without intergroup differences. To maintain comparable cardiac preload, the cumulative amount of colloid infusion was nonsignificantly higher in the control group (control group 270 [159;335], L-NIL group 200 [149;217] mL/kg, P = 0.194). Although the filling pressures were higher in the control animals, stroke volume tended to be higher in L-NIL-treated pigs (P = 0.015 at 18 h, P = 0.121 at 24 h). The increased cardiac output resulted in a significant rise of systemic oxygen delivery without intergroup difference, whereas systemic oxygen consumption did not change in either of the two groups over time.
Intestinal hemodynamics and metabolism
The data on intestinal perfusion, oxygen exchange, and metabolism are summarized in Table 2 as well as in Figures 1 and 2. The absolute values of superior mesenteric blood flow increased during the course of septic shock in both groups. However, the contribution of mesenteric blood flow to total cardiac output decreased over time in the control group. This blood flow distribution was not affected by the treatment with L-NIL. Both intestinal oxygen delivery and intestinal oxygen consumption remained unchanged during the experiment in both groups. Although the mesenteric blood flow was maintained, the continuous infusion of live bacteria progressively reduced ileal mucosal perfusion (251 [134;285] at baseline versus 78 [57;90] perfusion units at 24 h; Fig. 1). Selective iNOS inhibition significantly blunted these septic shock-induced changes in intestinal mucosal microcirculation (210 [181;277] at baseline versus 119 [102;140] perfusion units at 24 h), the intergroup difference being significant (P = 0.009) at the end of the experiment. Septic shock also resulted in a progressive significant increase in ileal mucosal-arterial PCO2 gap (Fig. 1) from a baseline value of 2.4 kPa (1.9;2.9) to 6.7 kPa (4.1;7.9) by the end of the experiment in animals challenged with P. aeruginosa only. By contrast, treatment with L-NIL reversed this _P. aeruginosa_-induced effect and restored the PCO2 gap back to the range of preshock values (2.2 [2.1;2.9] at baseline; 2.5 [1.5;3.4] at 24 h.). Although mesenteric venous pH markedly decreased from 7.43 (7.36;7.44) to 7.16 (7.10;7.29) in the control group, L-NIL prevented the gradual progression of mesenteric venous acidosis (Fig. 2). The corresponding values of mesenteric venous pH at baseline compared with 24 h in this group were 7.40 (7.36;7.41) and 7.30 (7.29;7.36), respectively. In the control group, the decreased mesenteric venous pH was associated with a rise in mesenteric venous lactate/pyruvate ratios by the end of the experiment, whereas no significant changes were observed over time in animals treated by L-NIL (Fig. 2).
Intestinal hemodynamic, oxygen exchange, and metabolic parameters
Ileal mucosal perfusion (top panel) and ileal mucosal-arterial PCO2 gap (bottom panel) in the CON (open plot, n = 8) and L-NIL (hatched plot, n = 8) groups. Data are median, 25%/75% quartiles, and 5th and 95th quantile range. *Designates significant difference versus baseline. §Designates significant difference in L-NIL versus CON.
Mesenteric venous lactate/pyruvate ratio (top panel) and mesenteric venous pH (bottom panel) in the CON (open plot, n = 8) and L-NIL (hatched plot, n = 8) groups. Data are median, 25%/75% quartiles, and 5th and 95th quantile range. *Designates significant difference versus baseline. §Designates significant difference in L-NIL versus CON.
Liver hemodynamics and metabolism
The data on liver perfusion, oxygen exchange and metabolism are summarized in Table 3 as well as in Figures 3 and 4. Due to the enhanced cardiac output, portal venous blood flow and, thereby, total liver blood flow increased significantly in both groups, without significant intergroup differences. Nevertheless, the tendency of total liver blood flow to increase was even more pronounced in the L-NIL group (P = 0.073 L-NIL vs. the control group at 24 h), mainly due to an increased hepatic arterial blood flow in the treatment group. When related to the changes in cardiac output, no significant blood flow redistributions were observed in both groups. Although the liver oxygen delivery was higher in the treatment group, there was no significant influence on hepatic oxygen consumption in either group. Infusion of P. aeruginosa induced a gradual fall in hepatic lactate uptake rates (Fig. 3). L-NIL treatment significantly mitigated this decline after 12 h of the treatment (P = 0.012 versus the control group). The decreased lactate uptake rate was accompanied by increased (P = 0.019) hepatic venous lactate/pyruvate ratio in the control group by the end of the experiment, whereas no significant increase was found in animals receiving L-NIL (Fig. 4). L-NIL also blunted the otherwise marked fall in hepatic venous ketone body ratio (Fig. 4), although the intergroup difference did not reach statistical significance (P = 0.053). Similarly, the progressive hepatic venous acidosis, which developed in response to P. aeruginosa, was markedly attenuated by L-NIL (Fig. 3).
Liver hemodynamic, oxygen exchange, and metabolic parameters
Liver lactate uptake rate (top panel) and hepatic venous pH (bottom panel) in the CON (open plot, n = 8) and L-NIL (hatched plot, n = 8) groups. Data are median, 25%/75% quartiles, and 5th and 95th quantile range. *Designates significant difference versus baseline. §Designates significant difference in L-NIL versus CON.
Hepatic venous lactate/pyruvate ratio (top panel) and hepatic venous ketone body ratio (bottom panel) in the CON (open plot, n = 8) and L-NIL (hatched plot, n = 8) groups. Data are median, 25%/75% quartiles, and 5th and 95th quantile range. *Designates significant difference versus baseline.
Hepatocellular and kidney injury
In the control group, serum concentrations of ALT increased over time (Fig. 5), demonstrating the development of liver injury. Treatment of pigs with L-NIL significantly blunted this rise in ALT. Likewise, L-NIL largely prevented the otherwise progressive rise in serum creatinine levels, a marker for the renal injury (Fig. 5).
Plasma levels of alanine aminotransferase (top panel) and creatinine (bottom panel) in the CON (open plot, n = 8) and L-NIL (hatched plot, n = 8) groups. Data are median, 25%/75% quartiles, and 5th and 95th quantile range. *Designates a significant difference versus baseline. §Designates a significant difference in L-NIL versus CON.
Arterial 8-isoprostane concentrations
P. aeruginosa infusion resulted in a significant gradual increase in 8-isoprostane plasma levels. Treatment with L-NIL significantly attenuated the formation of 8-isoprostane concentrations (P = 0.021 versus the control group; Fig. 6).
Arterial 8-isoprostane concentration in the CON (open plot, n = 8) and L-NIL (hatched plot, n = 8) groups. Data are median, 25%/75% quartiles, and 5th and 95th quantile range. *Designates a significant difference versus baseline. §Designates a significant difference in L-NIL versus CON.
DISCUSSION
We have recently demonstrated that selective iNOS inhibition with 1400W reversed the derangement of both intestinal and liver energy status caused by Escherichia coli lipopolysaccharide in a long-term, hyperdynamic porcine endotoxemia (12), contrasting with no beneficial effects of nonselective inhibition under these conditions (21, 22). In the current study, we tested the hypothesis that selective iNOS inhibition would be protective in the same model, where hyperdynamic sepsis is caused by a live P. aeruginosa. Hence, the present study largely extends our results, showing that selective iNOS inhibition is also protective when pigs are exposed to live bacteria challenge. Indeed, L-NIL restored blood pressure to baseline levels without reducing cardiac output or regional hemodynamics, and it significantly reduced the detrimental consequences of P. aeruginosa on intestinal and hepatocellular energy balance.
The great majority of studies investigating the role of NO-dependent pathways in the pathogenesis of sepsis and septic shock have used hypodynamic, unresuscitated rodent models challenged with a large doses of endotoxin (3). However, these models are criticized for their limited clinical relevance, and many emerging therapeutic approaches that have been found effective in these models failed to yield a benefit in larger animal models (11). By contrast, the sepsis model used in our study replicates many of the features of adequately resuscitated human septic shock, and substantial instrumentalization as used offers a broad insight into organ hemodynamic and metabolic pathways. It could be argued that our design is not only a model of bacterial sepsis, but also a model of postoperative sepsis induced by bacterial infusion. However, previous studies using a very similar surgical approach showed that there were no changes over 24 h of endotoxemia in all hemodynamic and metabolic parameters in the control sham-operated animals (23, 24).
In contrast to endotoxemic animal models, limited evidence is available to document a relationship between excessive NO production and cardiovascular as well as organ dysfunction in bacteremic models. Conflicting data have also been reported concerning the evidence for an overproduction of NO in nonrodent animal models of sepsis (25). In this context, we have previously shown that porcine endotoxemia is associated with an increased NO production: both increased total body nitrate pool and production rate (26) as well as increased exhaled amount of NO (12) could be demonstrated. These results are in agreement with recent findings from Bruins et al. (27), providing further evidence for increased NO production, particularly by the splanchnic organs during 24 h of hyperdynamic porcine endotoxemia. In the current study, we measured blood NOx levels as a surrogate marker for total NO production because direct quantification of iNOS activity was not possible. Progressive increase in the NOx concentrations suggests that live P. aeruginosa infusion caused prolonged NO production, a finding that is consistent with data reported previously (28). By contrast, treatment with L-NIL prevented this otherwise gradual rise in plasma NOx levels, indicating that this compound, which has 28-fold selectivity toward iNOS (19) and no effect on baseline mean arterial pressure (14), effectively reduced NO production.
As in our previous study, the administration of the iNOS inhibitor was started at the time of already well-established hyperdynamic circulation with concomitant signs of organ metabolic alterations that mimic the clinical situation. Consistent with those results, treatment with L-NIL prevented a _P. aeruginosa_-induced progressive fall in blood pressure without deteriorating cardiac output. Interestingly, despite comparable cardiac preload as documented by similar intrathoracic blood volume, pigs treated with L-NIL required lower cardiac filling pressures to maintain the same increase in stroke volume. Although the exact mechanism cannot be inferred from our study, it is tempting to assume that this compound improved myocardial compliance and exerted a positive inotropic effect. This finding corroborates previous reports demonstrating a beneficial effect of iNOS inhibition on myocardial performance in a porcine model of gram-positive shock (29), as well as in endotoxemic mice (30).
Treatment with L-NIL was associated with improved gut and liver metabolic capacity, and based on the data presented here, we cannot unambiguously determine the contribution of improved microcirculatory perfusion and/or restored mitochondrial respiration. In fact, _P. aeruginosa_-induced septic shock caused the progressive deterioration in mucosal microvascular perfusion as assessed by laser Doppler flowmetry, a finding supporting the view that the alterations in mucosal microcirculatory functions cannot be predicted from regional circulation (23). Nevertheless, L-NIL, albeit significantly, did not completely restore the ileal mucosal microvascular flow. This observation is in agreement with a study from Siegmund et al. (31) demonstrating that iNOS inhibition with 1400W normalized the ileal-mucosal PCO2 gradient together with a restoration of both the mucosal and the serosal microvascular PO2 in fluid-resuscitated porcine endotoxic shock. On the other hand, our results are in sharp contrast to data from Pittner et al. (28) who demonstrated that the mechanism of iNOS inhibition-associated prevention of ileal mucosal acidosis was not related to improved microcirculation during 24 h of porcine hyperdynamic endotoxemia. Although the exact explanation for this striking difference is not readily apparent, several possible explanations can be taken into consideration. First, despite aggressive fluid resuscitation infusion of live bacteria resulted in marked fall in arterial pressure, whereas animals in the aforementioned study exhibited normotensive hyperdynamic circulation. Because L-NIL prevented the hypotension, it is plausible to assume that L-NIL-mediated improvement in ileal mucosal microcirculation caused by increasing perfusion pressure to the intestine may have played a role in this context. It should be noted, however, that in the study by Pittner et al. (28), 1400W prevented the development of mucosal acidosis without affecting microvascular perfusion. Hence, merely restoring blood pressure is insufficient to explain the favorable effect of iNOS blockade on sepsis-induced metabolic disturbances. Second, the protective effects of iNOS inhibition on mucosal microcirculation seen in the present study were obtained with a selective iNOS inhibitor, which is chemically unrelated to the previously used 1400W. In fact, in a rat model of endotoxic shock, L-NIL not only reversed arterial hypotension but also restored the vasodilatory response to acetylcholine and sodium nitroprusside back to normal (14). Hence, it is tempting to speculate that an improved vasoregulation affiliated with restored vasomotoricity (32) and preserved endothelium-derived vasodilation (33) represents the underlying mechanism of iNOS blockade-mediated improvement of gut microvasculature.
Alternatively, the protective effects afforded by L-NIL may not be entirely related to the improved microcirculatory dysfunction. As L-NIL attenuated the otherwise progressive rise in both mesenteric and hepatic venous acidosis, prevented the alterations in regional cytosolic redox state, and blunted hepatic venous ketone body ratio, indicating defective oxidative energy metabolism, the mitigation of sepsis-associated derangement in intestinal and liver bioenergetic status may have also resulted from a favorable influence of iNOS blockade on mitochondrial respiration (34). Although L-NIL attenuated the changes in intestinal mucosal perfusion, the perfusion rate was still significantly less than the baseline value. In contrast, the ileal mucosal-arterial PCO2 gap was completely normal at 24 h in the L-NIL group. Taken together, these findings argue in support of the notion that a major effect of L-NIL on the ileal mucosa was related to a change in cellular biochemistry rather than just a change in microvascular perfusion. Although not directly addressed in our study, this concept is in accordance with a study by King et al. (35) showing that blocking iNOS activity with aminoguanidine prevented the reduction of cellular oxygen utilization in mucosal cell layer. These authors also demonstrated that iNOS inhibition ameliorated the endotoxin-induced depression of mitochondrial function (36). Moreover, the putative positive effect of iNOS inhibition on cytopathic hypoxia (37) is further supported by the results of a recent human study demonstrating that increased NO levels in muscle measured as NOx concentrations are associated with mitochondrial dysfunction, decreased ATP concentrations, organ failure, and eventual outcome (9).
The alterations in hepatosplanchnic energy metabolism induced in this study by live bacteria seem to be dependent on the NO-related pathways because treatment of pigs with the iNOS inhibitor largely ameliorated these changes. Moreover, similar protective effects have also been observed previously with a chemically dissimilar isoform-selective iNOS inhibitor 1400W, suggesting that the effects of both inhibitors were presumably related to the iNOS inhibition rather than some other nonspecific pharmacological properties. Interestingly, L-NIL substantially reduced the oxidative stress as documented by markedly attenuated increase in plasma isoprostane levels. This fact suggests that L-NIL-mediated protection was, at least in part, related to the restriction of the indirect cytotoxic pathways initiated by the toxic derivates of NO such as peroxynitrite (38) rather than the inhibition of NO itself. These results corroborate recently published data showing that iNOS inhibition with L-NIL reduced endotoxin-induced oxidative stress in rat liver (39).
In summary, microcirculatory, metabolic, and oxidative stress data from the present study indicate that the continuous infusion of the selective iNOS inhibitor L-NIL afforded significant positive benefit in the long-term model of porcine gram-negative, live bacteria-induced septic shock. These findings confirming our results from endotoxemic pigs reinforce the hypothesis that iNOS-generated NO-dependent pathways play a role in the pathophysiology of sepsis-induced metabolic changes that can contribute to organ dysfunction. Further investigation is necessary to determine the relative contribution of microvascular or/and cellular effects of the NO pathway in sepsis-induced organ dysfunction.
ACKNOWLEDGMENTS
The authors thank L. Trefil and J. Audesova for their skillfull technical assistance.
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Keywords:
Sepsis; septic shock; nitric oxide; nitric oxide synthase inhibitor; liver; gut; microcirculation; oxidative stress
©2004The Shock Society