Echocardiographic and survival studies in mice undergoing endotoxic shock: effects of genetic ablation of inducible nitric oxide synthase and pharmacologic antagonism of platelet-activating factor.

Evidence implicating inducible nitric oxide synthase (iNOS) in the alterations of cardiac function characteristic of septic shock has come mostly from studies on anesthetized animals, isolated hearts, cultured myocytes, or hosts treated with pharmacologic inhibitors that lack complete specificity for iNOS. Platelet-activating factor (PAF) can participate in the induction of iNOS and has also been implicated in cardiac dysfunction in sepsis. The present studies assessed cardiac function in a model of sepsis in awake mice in which the gene for iNOS was either normal or selectively disrupted. Mice of each genotype were treated with parenteral fluids or with a highly specific antagonist of PAF. Endotoxic shock was induced by challenge with bacterial lipopolysaccharide (LPS) after priming with heat-killed Propionobacterium acnes. Wild-type mice increased stroke volume and cardiac output in response to LPS. These changes were absent in iNOS-deficient mice. When treated with parenteral fluids, LPS-challenged wild-type and iNOS-deficient mice both had a marked reduction in cardiac output. Antagonism of PAF had no effect on echocardiographic indices in wild-type mice, but selectively overcame the bradycardia and reduced cardiac output elicited by fluid administration in LPS-shocked, iNOS-deficient mice. Thus, there are major cardiovascular effects of PAF that are shared by rather than mediated by iNOS. Neither complete iNOS deficiency nor antagonism of PAF improved survival, whether tested as single or combined intervention. On the contrary, complete deficiency of iNOS was detrimental to survival. Finally, we tested the hypothesis that iNOS deficiency might improve survival if the deficiency were specific but partial. For this, we used mice with one normal and one disrupted gene for iNOS. No survival advantage was evident for these iNOS heterozygotes. Thus, partial or complete inhibition of iNOS, with or without antagonism of PAF, afforded no evident benefit beyond the previously demonstrated reduction in hypotension. Finally, these studies demonstrate that echocardiography preceded by acclimatization is feasible in unanesthetized mice, a finding which should expand the value of genetically manipulated animals for analysis of cardiac function.

Lethality of endotoxin in mice genetically deficient in the respiratory burst oxidase, inducible nitric oxide synthase, or both.

Two classes of oxidants are thought to play a critical role in tissue damage in septic shock: reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). Particular importance has been ascribed to peroxynitrite, a product arising from the reaction of nitric oxide with superoxide. A major source of ROI is the respiratory burst oxidase of neutrophils, eosinophils, monocytes, and macrophages. A major source of RNI is inducible nitric oxide synthase (iNOS), an enzyme expressed in leukocytes, hepatocytes, vascular smooth muscle cells, endothelium, and cardiac myocytes during inflammation. In previous studies using various mouse models of endotoxic shock, genetic deficiency of iNOS as a sole intervention did not consistently alter survival. Here, using Salmonella typhimurium endotoxic bacterial lipopolysaccharide (LPS) as a sole challenge, genetic deficiency of iNOS was associated with no protection or a reduction in survival, depending on the dose of LPS. Further, no protection from lethality was observed when LPS was injected into mice genetically deficient in the 91 kDa subunit of the respiratory burst oxidase (gp91phox) nor in mice genetically deficient in both gp91phox and iNOS (gp91phox-/-/NOS2-/- mice). For the latter experiments, mice were challenged either with S. typhimurium LPS alone or with inactivated bacille Calmette-Guerin (BCG) followed by Escherichia coli LPS. Deficiency of gp91phox impaired the inflammatory response to inactivated Propionobacterium acnes, rendering survival studies following priming with P. acnes difficult to interpret. Thus, in two models of endotoxic shock, major reductions in the ability to form nitric oxide or superoxide, alone or in combination, failed to improve survival.

Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase.

The two genetically established antimicrobial mechanisms of macrophages are production of reactive oxygen intermediates by phagocyte oxidase (phox) and reactive nitrogen intermediates by inducible nitric oxide synthase (NOS2). Mice doubly deficient in both enzymes (gp91(phox-/-)/NOS2(-/-)) formed massive abscesses containing commensal organisms, mostly enteric bacteria, even when reared under specific pathogen-free conditions with antibiotics. Neither parental strain showed such infections. Thus, phox and NOS2 appear to compensate for each other's deficiency in providing resistance to indigenous bacteria, and no other pathway does so fully. Macrophages from gp91(phox-/-)/NOS2(-/-) mice could not kill virulent Listeria. Their killing of S. typhimurium, E. coli, and attenuated Listeria was markedly diminished but demonstrable, establishing the existence of a mechanism of macrophage antibacterial activity independent of phox and NOS2.