Time course of nitric oxide synthases, nitrosative stress, and poly(ADP ribosylation) in an ovine sepsis model

Abstract

IntroductionDifferent isoforms of nitric oxide synthases (NOS) and determinants of oxidative/nitrosative stress play important roles in the pathophysiology of pulmonary dysfunction induced by acute lung injury (ALI) and sepsis. However, the time changes of these pathogenic factors are largely undetermined.MethodsTwenty-four chronically instrumented sheep were subjected to inhalation of 48 breaths of cotton smoke and instillation of live Pseudomonas aeruginosa into both lungs and were euthanized at 4, 8, 12, 18, and 24 hours post-injury. Additional sheep received sham injury and were euthanized after 24 hrs (control). All animals were mechanically ventilated and fluid resuscitated. Lung tissue was obtained at the respective time points for the measurement of neuronal, endothelial, and inducible NOS (nNOS, eNOS, iNOS) mRNA and their protein expression, calcium-dependent and -independent NOS activity, 3-nitrotyrosine (3-NT), and poly(ADP-ribose) (PAR) protein expression.ResultsThe injury induced severe pulmonary dysfunction as indicated by a progressive decline in oxygenation index and concomitant increase in pulmonary shunt fraction. These changes were associated with an early and transient increase in eNOS and an early and profound increase in iNOS expression, while expression of nNOS remained unchanged. Both 3-NT, a marker of protein nitration, and PAR, an indicator of DNA damage, increased early but only transiently.ConclusionsIdentification of the time course of the described pathogenetic factors provides important additional information on the pulmonary response to ALI and sepsis in the ovine model. This information may be crucial for future studies, especially when considering the timing of novel treatment strategies including selective inhibition of NOS isoforms, modulation of peroxynitrite, and PARP.

Administration of tetrahydrobiopterin improves the microcirculation and outcome in an ovine model of septic shock*

“…In a peritonitis-induced ovine septic shock model, Su et al (30) showed that the selective iNOS inhibitor, BYK191023, blunted the increase in pulmonary artery pressure and improved gas exchange, microcirculation, and tissue metabolism. Similar findings with selective iNOS inhibition were observed in an ovine sepsis model induced by smoke inhalation with lung installation of bacteria (40,41) and in a porcine bacteremia model (42). These studies indicate that iNOS inhibition may exert its positive effects by reducing eNOS overproduction.…”

Time course of the inflammatory and oxidative stress response to pulmonary infection in mice

“…Therefore, we now utilized the corresponding mouse model of pulmonary infection to compensate for this limitation. However, we found that the time course of protein contents of inflammatory cytokines in lung tissue in mice was similar to the time course of IL-8 protein content in sheep in the previous study [17]. This finding further suggests that the injuries in both animal models trigger similar pathophysiological reactions in the lung.…”

“…We have previously described the time changes in NO synthases, nitrosative stress, and poly(ADP ribosylation) in an ovine model of pulmonary sepsis [17]. Unfortunately, in this study, we were not able to measure cytokine concentrations, other than IL-8, because most available antibodies for biomolecular measurements do not cross-react with sheep tissue.…”

“…In addition, these studies investigated only a few time points during the initial phase after the onset of infection [12][13][14][15][16]. However, recent experimental evidence suggests that the initial phase may be critical [4,17].…”

Selective V1aagonism attenuates vascular dysfunction and fluid accumulation in ovine severe sepsis

“…Key mechanisms of vascular dysfunction include the activation of neutrophils and the subsequent interaction with the endothelium (diapedesis, "rolling and sticking") (20) as well as the increased production of nitric oxide via stimulation of the inducible nitric oxide synthase and reactive nitrogen species, such as peroxynitrite (22). In addition, mediators released by the endothelial cells, like VEGF and angiopoietin-2, contribute to the development of vascular leakage (25,32).…”