Soheyl Bahrami

Nitrite as Regulator of Hypoxic Signaling in Mammalian Physiology

In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade was thought to be irreversible under physiological conditions. However, a growing body of experimental observations attests that the presence of endogenous nitrite regulates a number of signaling events along the physiological and pathophysiological oxygen gradient. Hypoxic signaling events include vasodilation, modulation of mitochondrial respiration, and cytoprotection following ischemic insult. These phenomena are attributed to the reduction of nitrite anions to nitric oxide if local oxygen levels in tissues decrease. Recent research identified a growing list of enzymatic and nonenzymatic pathways for this endogenous reduction of nitrite. Additional direct signaling events not involving free nitric oxide are proposed. We here discuss the mechanisms and properties of these various pathways and the role played by the local concentration of free oxygen in the affected tissue.

Kinetics of endotoxin and tumor necrosis factor appearance in portal and systemic circulation after hemorrhagic shock in rats.

ObjectiveThis study was performed to investigate gut-derived bacterial translocation and the time course of endotoxin (lipopolysaccharide [LPS]) and tumor necrosis factor (TNF) appearance, both in portal and systemic circulation. Summary Background DataThe significance of intestinal bacteria/endotoxin translocation or TNF formation in the development of systemic sepsis has been disputed. MethodsA rat model of hemorrhagic shock (30–35 mm Hg for 90 min) and resuscitation was used. ResultsBacterial translocation was histologically observed in the small intestinal wall 30 minutes after resuscitation. A significant increase in LPS concentrations was found in the portal vein (91.7 ± 30.6 pg/mL) at 90 minutes, which remained steady until 150 minutes after shock.Lipopolysaccharide increased in the systemic circulation, the levels became significant at 120 minutes, and peaked (66.5 ± 39.2 pg/mL) 150 minutes after shock. Tumor necrosis factor concentrations were found to be significantly elevated in both portal and systemic circulation (75.6 ± 22.1 vs. 58.4 ± 14.1 pg/mL) at 90 minutes post-shock. Although there was no further increase in TNF concentration in the portal blood, TNF peaked (83.5 ± 17.7 pg/mL) in systemic circulation at 120 minutes and still was markedly increased at 150 minutes post-shock. In addition, higher LPS and TNF concentrations in systemic circulation were found in the nonsurvivors than in the surviving animals at the end of resuscitation. ConclusionsThese results suggest that hemorrhagic shock may lead to early bacterial translocation in the intestinal wall and transient access of gut-derived LPS and LPS-induced mediators into the circulation predominantly via the portal circulation.

Clinical detection of LPS and animal models of endotoxemia

The interest in the study of endotoxemia in the clinical area has increased recently as a result of a) improved and simplified endotoxin determination e.g. chromogenic-kinetic microplate methods (also an improved blood sampling tool is available), b) incidence of sepsis has increased due to improvement in early (e.g. posttraumatic) survival, c) interest in and good evidence for gut translocation as a source of endotoxemia, d) agents have developed, which can antagonize endotoxins. There is evidence that patients with positive endotoxin test in the ICU have a higher incidence of organ failure. To study the pathophysiological consequences of endotoxemia and possible ways of intervention animal models are necessary. The choice of the experimental setting depends on the aim of the study e.g. whether prolonged observation is necessary in survival studies or whether hemodynamic variables have to be measured or whether therapeutic agents only crossreact with primates. Since LPS levels are quite low in clinical studies, an important factor for selection of a relevant animal might be LPS sensitivity, or the use of additional sensitization techniques e.g. galactosamine. Another important aspect in this context is whether LPS is given as bolus or infused up to several days. In this review the dose, time, and route of LPS administration is also discussed. For screening purposes rodents are usually used, or sometimes rabbits due to their higher LPS sensitivity. Another very sensitive animal model is the sheep, which can be chronically instrumented and as a specialty allows lung lymph drainage and thus studies of LPS effects on pulmonary permeability. Pigs are used for hemodynamic studies and often in therapeutical studies if species-specificity of the drug tested is not important, in cases where a large animal is necessary. Finally the non-human primates offer a number of advantages due to human-like physiology, due to the cross-reactivity of human assay systems and accordingly also cross-reactivity of human therapeutic agents. While the chimpanzee also shares the LPS sensitivity of humans, baboons are insensitive like rodents. Thus each model serves to provide some useful purpose and the selection must be made to meet the requirements of the specific questions to be asked, with special emphasis of the chosen endotoxin model on relevance for the human sepsis state.

Thromboelastometric (ROTEM) findings in patients suffering from isolated severe traumatic brain injury.

Severe traumatic brain injury (sTBI) is often accompanied by coagulopathy and an increased risk of bleeding. To identify and successfully treat bleeding disorders associated with sTBI, rapid assessment of coagulation status is crucial. This retrospective study was designed to assess the potential role of whole-blood thromboelastometry (ROTEM(®), Tem International, Munich, Germany) in patients with isolated sTBI (abbreviated injury scale [AIS](head) ≥3 and AIS(extracranial) <3). Blood samples were obtained immediately following admission to the emergency room of the Trauma Centre Salzburg in Austria. ROTEM analysis (EXTEM, INTEM, and FIBTEM tests) and standard laboratory coagulation tests (prothrombin time index [PTI, percentage of normal prothrombin time], activated partial thromboplastin time [aPTT], fibrinogen concentration, and platelet count) were compared between survivors and non-survivors. Out of 88 patients with sTBI enrolled in the study, 66 survived and 22 died. PTI, fibrinogen, and platelet count were significantly higher in survivors (p<0.005). Accordingly, aPTT was shorter in this group (p<0.0001). ROTEM analysis revealed shorter clotting times in extrinsically activated thromboelastometric test (EXTEM) and intrinsically activated thromboelastometric test (INTEM) (p<0.001), shorter clot formation times in EXTEM and INTEM (p<0.0001), and higher maximum clot firmness in EXTEM, INTEM, and FIBTEM (p<0.01) in survivors compared with non-survivors. Logistic regression analysis revealed extrinsically activated thromboelastometric test with cytochalasin D (FIBTEM) MCF and aPTT to have the best predictive value for mortality. According to the degree of coagulopathy, non-survivors received more RBC (p=0.016), fibrinogen concentrate (p=0.01), and prothrombin complex concentrate (p<0.001) within 24 h of arrival in the emergency room. ROTEM testing appeared to offer an early signal of severe life-threatening sTBI. Further studies are warranted to confirm these results and to investigate the role of ROTEM in guiding coagulation therapy.

Pathogenesis of hemorrhage-induced bacteria/endotoxin translocation in rats. Effects of recombinant bactericidal/permeability-increasing protein.

ObjectiveThis study was conducted to determine the role of gut-derived bacteria/endotoxin in the pathogenesis of the multiple-organ damage and mortality, the possible beneficial effect of recombinant bactericidal/permeability-increasing protein (rBPI21), and whether neutralizing endotoxemia by rBPI21 treatment influences tumor necrosis factor (TNF) formation in rats after hemorrhagic shock and resuscitation. Summary Background DataHypovolemic shock might be associated with bacterial or endotoxin translocation as well as systemic sepsis. Similar to bactericidal/permeability-increasing (BPI) protein, rBPI21 has been found to bind endotoxin and inhibit TNF production. MethodsA rat model of prolonged hemorrhagic shock (30 to 35 mm Hg for 180 min) followed by adequate resuscitation was employed. Recombinant bactericidal/permeability-increasing protein was administered at 5 mg/kg intravenously. The control group was treated similarly to the BPI group, but received thaumatin as a protein-control preparation in the same dose as rBPI21. ResultsImmediately after resuscitation (230 min), plasma endotoxin levels in the control group (61.0 ± 16.3 pg/mL) were almost neutralized by rBPI21 treatment (13.8 ± 4.8 pg/mL, p < 0.05). Plasma TNF levels were not significantly influenced by rBPI21 treatment. The 48-hour survival rate was 68.8% in the treatment group versus 37.5% in the control group (p = 0.08). Microscopic histopathologic examination revealed relatively minor damage to various organs in the treatment group. ConclusionsThese data suggest that hemorrhagic shock may lead to bacterial/endotoxin translocation with concomitant TNF formation, endogenous endotoxemia may play an important role in the pathogenesis of multiple-organ failure after shock and trauma, TNF formation at an early stage

ABANDON THE MOUSE RESEARCH SHIP? NOT JUST YET!

ABSTRACT Many preclinical studies in critical care medicine and related disciplines rely on hypothesis-driven research in mice. The underlying premise posits that mice sufficiently emulate numerous pathophysiologic alterations produced by trauma/sepsis and can serve as an experimental platform for answering clinically relevant questions. Recently, the lay press severely criticized the translational relevance of mouse models in critical care medicine. A series of provocative editorials were elicited by a highly publicized research report in the Proceedings of the National Academy of Sciences (PNAS; February 2013), which identified an unrecognized gene expression profile mismatch between human and murine leukocytes following burn/trauma/endotoxemia. Based on their data, the authors concluded that mouse models of trauma/inflammation are unsuitable for studying corresponding human conditions. We believe this conclusion was not justified. In conjunction with resulting negative commentary in the popular press, it can seriously jeopardize future basic research in critical care medicine. We will address some limitations of that PNAS report to provide a framework for discussing its conclusions and attempt to present a balanced summary of strengths/weaknesses of use of mouse models. While many investigators agree that animal research is a central component for improved patient outcomes, it is important to acknowledge known limitations in clinical translation from mouse to man. The scientific community is responsible to discuss valid limitations without overinterpretation. Hopefully, a balanced view of the strengths/weaknesses of using animals for trauma/endotoxemia/critical care research will not result in hasty discount of the clear need for using animals to advance treatment of critically ill patients.

Nonspecific increase of systemic neuron-specific enolase after trauma: clinical and experimental findings.

The aim of this clinical and experimental study was to determine whether systemic neuron-specific enolase (NSE) is a useful early marker of traumatic brain injury (TBI) and whether NSE is affected by ischemia/reperfusion damage of abdominal organs. Our study included patients with and without TBI (verified by computerized tomography) admitted within 6 h after trauma and male Sprague-Dawley rats with ischemia and reperfusion of the abdominal organs liver, gut, or kidney. Thirty-eight study patients included 13 with isolated TBI and 18 patients with multiple trauma and TBI. Seven patients had multiple trauma but no TBI. Fifteen rats were anaesthetized and subjected to isolated ischemia of the liver, gut, or kidney (n = 5 each) for 1 h, followed by reperfusion for 3 h. In patients, NSE increased over 2-fold versus the upper normal limit (10 μg/L) within 6 h after trauma, regardless of whether TBI had occurred or not. In rats, NSE increased over 3-fold versus laboratory controls during ischemia of the liver and kidney (both P < 0.0005), but not of the gut. NSE increased over 2-fold after onset of reperfusion of the liver and kidney (both P < 0.05), but not of the gut and increased over 3-fold after 3 h of reperfusion of the liver, gut (both P < 0.005), and kidney (P < 0.0005). Our data show that systemic NSE increases to similar degrees with and without TBI. Therefore, NSE is not a useful early marker of TBI in multiple trauma.

Neutrophil function and lipid peroxidation in a rat model of multiple organ failure

Multiple organ failure (MOF) was induced by sterile intraperitoneal inoculation of zymosan in the rat. This results in a typical triphasic illness with maximal clinical signs at Days 2 and 14. In this study, granulocyte superoxide production (unstimulated and phorbol myristic acid stimulated) was studied as well as lipid peroxidation (TBAR) in plasma, liver, and lung tissue. Mainly TBAR levels in liver and lung tissue closely correlated with the triphasic clinical illness, while bacteriological data did not. It is concluded that the severe inflammatory response in this experimental model probably is the result of excessive toxic oxygen radical production. The first phase of illness may mainly be due to oxygen radical formation by activated PMN, the third phase of illness to the production of lysosomal enzymes (proteinases) from PMN, and activated macrophages as indicated by elevated N-acetylglucosaminidase levels.

Epr analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species

The excessive formation of reactive oxygen and nitrogen species (RONS) in tissue has been implicated in the development of various diseases. In this study we adopted ex vivo low temperature EPR spectroscopy combined with spin trapping technique to measure local RONS levels in frozen tissue samples. CP-H (1-hydroxy-3-carboxy-pyrrolidine), a new nontoxic spin probe, was used to analyze RONS in vivo. In addition, nitrosyl complexes of hemoglobin were determined to trace nitric oxide released into blood. By this technique we found that RONS formation in tissue of control animals increased in the following order: liver < heart < brain < cerebellum < lung < muscle < blood < ileum < kidney < duodenum < jejunum. We also found that endotoxin challenge, which represents the most common model of septic shock, increased the formation of RONS in rat liver, heart, lung, and blood, but decreased RONS formation in jejunum. We did not find changes in RONS levels in other parts of gut, brain, skeletal muscles, and kidney. Scavenging of RONS by CP-H was accompanied by an increase in blood pressure, indicating that LPS-induced vasodilatation may be due to RONS, but not due to nitric oxide. Experiments with tissue homogenates incubated in vitro with CP-H showed that ONOO(-) and O(2)(*)(-), as well as other not identified RONS, are detectable by CP-H in tissue. In summary, low-temperature EPR combined with CP-H infusion allowed detection of local RONS formation in tissues. Increased formation of RONS in response to endotoxin challenge is organ specific.

Small-volume fluid resuscitation with hypertonic saline prevents inflammation but not mortality in a rat model of hemorrhagic shock.

ABSTRACT Hemorrhage remains a primary cause of death in civilian and military trauma. Permissive hypotensive resuscitation is a possible approach to reduce bleeding in patients until they can be stabilized in an appropriate hospital setting. Small-volume resuscitation with hypertonic saline (HS) is of particular interest because it allows one to modulate the inflammatory response to hemorrhage and trauma. Here, we tested the utility of permissive hypotensive resuscitation with hypertonic fluids in a rat model of hemorrhagic shock. Animals were subjected to massive hemorrhage [mean arterial pressure (MAP) = 30 - 35 mmHg for 2 h until decompensation] and partially resuscitated with a bolus dose of 4 mL/kg of 7.5% NaCl (HS), hypertonic hydroxyl ethyl starch (HHES; hydroxyl ethyl starch + 7.5% NaCl), or normal saline (NS) followed by additional infusion of Ringer solution to maintain MAP at 40 to 45 mmHg for 40 min (hypotensive state). Finally, animals were fully resuscitated with Ringer solution and the heparinized shed blood. Hypotensive resuscitation with NS caused a significant increase in plasma interleukin (IL)-1&bgr;, IL-6, IL-2, interferon &ggr; (IFN&ggr;), IL-10, and granulocyte-macrophage colony stimulating factor (GM-CSF). This increase was blocked by treatment with HS. HHES treatment significantly reduced the increase of IL-1&bgr; and IL-2 but not that of the other cytokines studied. Despite the strong effects of HS and HHES on cytokine production, both treatments had little effect on plasma lactate, base excess (BE), white blood cell (WBC) count, myeloperoxidase (MPO) content, and the wet/dry weight ratio of the lungs. Moreover, on day 7 after shock, the survival rate in rats treated with HS was markedly, but not significantly, lower than that of NS-treated animals (47% vs. 63%, respectively). In summary, hypotensive resuscitation with hypertonic fluids reduces the inflammatory response but not lung tissue damage or mortality after severe hemorrhagic shock.