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Background. Organ injury and dysfunction in sepsis accounts for significant morbidity and mortality. Adaptive cellular responses in the setting of sepsis prevent injury and allow for organ recovery. We and others have shown that part of the adaptive response includes regulation of cellular respiration and maintenance of a healthy mitochondrial population. Herein, we hypothesized that endotoxin-induced changes in hepatocyte mitochondrial respiration and homeostasis are regulated by an inducible nitric oxide synthase/nitric oxide (iNOS/NO)-mitochondrial reactive oxygen species (mtROS) signaling axis, involving activation of the NRF2 signaling pathway.Methods. Wild-type (C57Bl/6) or iNos-/-male mice were subjected to intraperitoneal lipopolysaccharide (LPS) injections to simulate endotoxemia. Individual mice were randomized to treatment with NO-releasing agent DPTA-NONOate, mtROS scavenger MitoTEMPO, or vehicle controls. Other mice were treated with scramble orNrf2-specific siRNAviatail vein injection. Primary murine hepatocytes were utilized forin vitrostudies with or without LPS stimulation. Oxygen consumption rates were measured to establish mitochondrial respiratory parameters. Western blotting, confocal microscopy with immunocytochemistry, and rtPCR were performed for analysis of iNOS as well as markers of both autophagy and mitochondrial biogenesis.Results. LPS treatment inhibited aerobic respirationin vitroin wild-type but notiNos-/-cells. Experimental endotoxemiain vivoorin vitroinduced iNOS protein and mtROS production. However, induction of mtROS was dependent on iNOS expression. Furthermore, LPS-induced hepatic autophagy/mitophagy and mitochondrial biogenesis were significantly attenuated iniNos-/-mice or cells with NO or mtROS scavenging. These responses were rescued iniNos-/-miceviadelivery of NO bothin vivoandin vitro. Conclusions. These data suggest that regulation of mitochondrial quality control following hepatocyte LPS exposure is dependent at least in part on a NO-mtROS signaling network. Further investigation to identify specific agents that modulate this process may facilitate the prevention of organ injury in sepsis.

Organ failure in sepsis accounts for significant mortality worldwide. Mitochondrial and metabolic responses are central to the overall response of the cell, and thus of the organ and organism. Adaptive responses in metabolism are critical to the recovery at the cellular level. The purpose of these investigations was to test the hypothesis that sepsis is associated with decreased aerobic respiration and significant metabolic changes in the liver.C57BL/6 mice underwent cecal ligation and puncture (CLP) with a 21 gauge needle or an operation without CLP. Mice were euthanized from 0-24 h after the procedure and liver tissue was harvested. Tissue oxygen consumption and mitochondrial complex activity were measured. Global biochemical profiles of 311 metabolites were performed at the 8-h time point (n = 8/group) and analyzed by gas chromatography-mass spectrometry and liquid chromatography tandem mass spectrometry platforms by Metabolon (Durham, North Carolina). The influence of lipopolysaccharide (LPS) on aerobic and anaerobic respiration in primary mouse hepatocytes was also investigated.CLP in vivo or LPS in vitro resulted in a significant decrease in hepatic oxygen consumption. There was a significant decrease in oxidative phosphorylation measured at 12 h. LPS also resulted in a significant increase in anaerobic respiration in hepatocytes. Interestingly, the metabolomic analysis resulted in a metabolic shift in the liver from carbohydrate-based energy to utilization of fatty acids and amino acids. This included an increase in every tricarboxylic acid cycle intermediate and derivative, suggesting an increased flux into the cycle from fatty acid beta-oxidation and anaplerotic contributions from amino acids.Sepsis results in a metabolic response and profile consistent with increased anaerobic respiration, which occurs prior to significant changes in hemodynamics. The metabolic responses of cells and organs may be important adaptive responses to prevent organ failure and death.

Objective The cellular injury that occurs in the setting of hemorrhagic shock and resuscitation (HS/R) affects all tissue types and can drive altered inflammatory responses. Resuscitative adjuncts hold the promise of decreasing such injury. Here we test the hypothesis that sodium nitrite (NaNO 2 ), delivered as a nebulized solution via an inhalational route, protects against injury and inflammation from HS/R. Methods Mice underwent HS/R to a mean arterial pressure (MAP) of 20 or 25 mmHg. Mice were resuscitated with Lactated Ringers after 90–120 min of hypotension. Mice were randomized to receive nebulized NaNO 2 via a flow through chamber (30 mg in 5 mL PBS). Pigs (30–35 kg) were anesthetized and bled to a MAP of 30–40 mmHg for 90 min, randomized to receive NaNO 2 (11 mg in 2.5 mL PBS) nebulized into the ventilator circuit starting 60 min into the hypotensive period, followed by initial resuscitation with Hextend. Pigs had ongoing resuscitation and support for up to four hours. Hemodynamic data were collected continuously. Results NaNO 2 limited organ injury and inflammation in murine hemorrhagic shock. A nitrate/nitrite depleted diet exacerbated organ injury, as well as mortality, and inhaled NaNO 2 significantly reversed this effect. Furthermore, NaNO 2 limited mitochondrial oxidant injury. In porcine HS/R, NaNO 2 had no significant influence on shock induced hemodynamics. NaNO 2 limited hypoxia/reoxia or HS/R-induced mitochondrial injury and promoted mitochondrial fusion. Conclusion NaNO 2 may be a useful adjunct to shock resuscitation based on its limitation of mitochondrial injury.

Aims Currently, there is no effective resuscitative adjunct to fluid and blood products to limit tissue injury for traumatic hemorrhagic shock. The objective of this study was to investigate the role of inhaled carbon monoxide (CO) to limit inflammation and tissue injury, and specifically mitochondrial damage, in experimental models of hemorrhage and resuscitation. Results Inhaled CO (250 ppm for 30 minutes) protected against mortality in severe murine hemorrhagic shock and resuscitation (HS/R) (20% vs. 80%; P

Background Mortality in sepsis is most often attributed to the development of multiple organ failure. In sepsis, inflammation-mediated endothelial activation, defined as a proinflammatory and procoagulant state of the endothelial cells, has been associated with severity of disease. Thus, the objective of this study was to test the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation limits inflammation and endothelium activation to protect against organ injury in sepsis. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is an adenosine monophosphate analog, has been used to upregulate activity of AMPK. Compound C is a cell-permeable pyrrazolopyrimidine compound that inhibits AMPK activity. Methods Wild-type mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice were randomized to vehicle, AICAR, or compound C. Mouse kidney endothelial cells were used for in vitro experiments. Renal and liver function were determined by serum cystatin C, blood urea nitrogen (BUN), creatinine, and alanine aminotransferase. Serum cytokines were measured by enzyme-linked immunosorbent assay. Microvascular injury was determined using Evans blue dye and electron microscopy. Immunohistochemistry was used to measure protein levels of phospho-AMPK (p-AMPK), microtubule-associated protein 1A/1B-light chain 3 (LC3), and intracellular adhesion molecule. LC3 levels were used as a measure of autophagosome formation. Results AICAR decreased liver and kidney injury induced by CLP and minimized cytokine elevation in vivo and in vitro. CLP increased renal and hepatic phosphorylation of AMPK and autophagic signaling as determined by LC3. Inhibition of AMPK with compound C prevented CLP-induced autophagy and exacerbated tissue injury. Additionally, CLP led to endothelial injury as determined by electron microscopy and Evans blue dye extravasation, and AICAR limited this injury. Furthermore, AICAR limited CLP and lipopolysaccharide (LPS)-induced upregulation of intracellular adhesion molecule in vivo and in vitro and decreased LPS-induced neutrophil adhesion in vitro. Conclusions In this model, activation of AMPK was protective, and AICAR minimized organ injury by decreasing inflammatory cytokines and endothelial activation. These data suggest that AMPK signaling influences sepsis or LPS-induced endothelial activation and organ injury.

The role of molecular genetics and genomics has risen to great importance in modern medicine. Medicine has seen an exponential growth in methods used to diagnose genetic aberrations leading to better understanding of disease processes. Examples of the advancement have impacted all medical and surgical specialties including improved prenatal screening for rare diseases, pharmacogenetics, and genetic profiling of malignancies. Improved understanding of disease processes at the genetic level has led to improvements in diagnosis, prognosis, and in some cases treatment. Advancements in laboratory methods for RNA and DNA analysis and sequencing have made possible our current understanding of both simple and complex pathology at its fundamental level. Surgeons, physicians, and healthcare professionals must develop an understanding of the methods of RNA and DNA analysis. Reviewed here are the basics and advancements in genetic and genomic analysis that are used in research and clinical medicine.