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Background: Nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS or NOS2) has been implicated in the hypotension, organ failure, and death that complicate sepsis. To avoid the confounding effects and limitations of iNOS inhibitors, we used iNOS gene "knockout" mice to examine the effect of inducible NO production in a model of polymicrobial abdominal sepsis treated with antibiotics. We hypothesized that iNOS gene deficiency would significantly alter outcome., Methods: C57BL6 wild-type (control) and congenic iNOS knockout mice were studied concurrently. Under halothane anesthesia, the ceca were ligated with 4-0 silk suture and punctured twice with a 26-gauge needle (cecal ligation and puncture, CLP). Survival was followed for 7 days, after which necropsies were performed in surviving animals. In an accompanying study examining the acute effects of sepsis, organ injury at 18 hours after CLP as determined by histology and the degree of cell death by apoptosis were examined with the use of hematoxylin and eosin (H&E) and TUNEL staining and two-channel fluorescence-activated cell sorter (FACS) analysis., Results: Sham laparotomy produced no lethality in either knockout (n = 3) or wild-type (n = 3) animals. Compared with survival in controls (n = 20), survival after CLP in iNOS knockout mice (n = 21) was significantly decreased (P < .01 at 2 days, P = .080 at 7 days, Mantel-Haenszel log-rank test). CLP-induced apoptotic cell death was significantly less in the thymus of iNOS knockout mice compared with wild-type mice., Conclusions: We conclude that iNOS gene function provides a survival benefit in septic mice and is associated with increased sepsis-induced thymocyte apoptosis. To our knowledge, this is the first survival study examining the effect of iNOS gene deficiency in a clinically relevant model of sepsis.

Background: Apoptosis, a mechanism of cell death prominent in critical illnesses including disseminated inflammation and multiorgan dysfunction syndrome, is characterized by morphologic changes including cell shrinkage, condensation of organelles, blebbing, and chromatin fragmentation. These phenomena suggest substantial changes in cytoskeletal structure. We hypothesized that stress-induced apoptosis in endothelial cells is, in part, a consequence of a critical cytoskeletal rearrangement., Methods: Porcine aortic endothelial cells in culture, surrogates for the microvasculature in vivo, were exposed sequentially to Escherichia coli endotoxin (25 micrograms/mL; 18 hours) to induce the inflammatory response and then to sodium arsenite (160 mumol/L; 120 minutes) to induce the heat-shock response, a well-characterized model of stress-induced apoptosis. Laser confocal micrographs of fluorescein isothio-cyanate-labeled phalloidin-stained cells were analyzed to calculate the border fractal dimension of the cytoskeleton. Other cells were exposed to cytochalasin D, a fungal metabolite, which interferes with polymerization of actin from its globular to its filamentous form, and similarly were analyzed with respect to fractal dimension, viability (neutral red assay), and manner of death (annexin V fluorescence-activated cell scanning analysis)., Results: Induction of the inflammatory or heat-shock responses caused subtle and distinct rearrangement of the actin cytoskeleton. When these stimuli were applied in sequence, a synergistic interaction led to profound cytoskeletal collapse. Reversal of the sequence did not induce the cytoskeletal disruption. Cytochalasin D alone induced a dose-dependent cytoskeletal collapse indistinguishable from that caused by the acute phase-heat shock sequence that caused cell death by apoptosis. The effect of lower doses of Cytochalasin D could be potentiated by subsequent induction of the heat-shock response., Conclusions: Sequential stresses that mimic pathophysiologic "two-hit" stimuli induce a characteristic fractal rearrangement of the actin cytoskeleton. Because cytochalasin D-induced rearrangement of this cytoskeleton produced apoptosis indistinguishable from the stress-induced apoptosis, we conclude that the cytoskeletal rearrangement is likely a critical event in the pathway to apoptosis. This disruption of intracellular interconnections mirrors endotoxin-induced disruption in signals among organs and supports the mechanistic hypothesis that multiorgan dysfunction syndrome generally reflects disruption of signals and connections at several levels of biologic organization.

Background: Iron participates in diverse pathologic processes by way of the Fenton reaction, which catalyzes the formation of reactive oxygen species (ROS). To test the hypothesis that this reaction accelerates apoptosis, we used human umbilical vein endothelial cells (HUVECs) as surrogates for the microvasculature in vivo., Methods: HUVECs were loaded with Fe [III](ferric chloride and ferric ammonium citrate) with 8-hydroxyquinoline as carrier and were then challenged with two stimuli of the heat shock response, authentic heat or sodium arsenite. Iron dependence was tested with two chelators, membrane-impermeable deferoxamine and membrane-permeable o-phenanthroline. The role of ROS was assessed with superoxide dismutase, catalase, and the reporter compound dichlorofluorescein diacetate. The mechanism of cell death was assessed with three complementary techniques, Annexin V/propidium iodide labeling, the TUNEL stain, and electron microscopy., Results: Iron-loaded HUVECs executed apoptosis after a heat shock stimulus. Iron-catalyzed formation of ROS appeared to be a critical mechanism, because both chelation of iron and enzymatic detoxification of ROS attenuated this apoptosis., Conclusions: Inorganic iron, in concert with chemical and physical inducers of the heat shock response, may trigger apoptosis. The accumulation of iron in injured tissue may thereby predispose to accelerated apoptosis and account, in part, for poor wound healing and organ failure.

Injury is attended by accelerated skeletal muscle proteolysis. Accurate definition of this hypercatabolic response and its mediation is requisite for specific therapy. We measured protein dynamics in the incubated and intact epitrochlearis and soleus muscles excised from both forelimbs and both hindlimbs of rats 4 days after injury by either a single hind limb scald (90 degrees C water for 3 seconds; metabolic rate (MR) + 15%, urinary urea nitrogen (UUN) + 10%) or a 5% excision (dorsal skin removed to fascia; MR + 40%, UUN + 90%). Protein synthesis (3H phenylalanine incorporation) increased only in the injured soleus from the scalded hind limb (+100%). Actin and myosin breakdown (3-methylhistidine release) increased in all muscles tested and was consistently larger in epitrochlearis than in soleus muscles. Breakdown of the mixed protein pool (tyrosine release) increased but less so than 3-methylhistidine and did not reach significance in the uninjured soleus muscle of scalded rats. With respect to fiber type, white fiber epitrochlearis muscle demonstrated a more pronounced elevation of both measures of breakdown but at a lower metabolic rate than did red fiber soleus muscle. Increasing MR was associated with a linear increase in soleus proteolysis but no further change in epitrochlearis breakdown. We conclude that protein breakdown is increased in skeletal muscle distant from injury; however, even when metabolic stress is severe, synthesis is unchanged. Muscles of different fiber composition are not equally labile. Furthermore, myofibrillar protein is more labile than the mixed protein pool.