Oscar McCook

Cardiopulmonary, histologic, and inflammatory effects of intravenous Na2S after blunt chest trauma-induced lung contusion in mice.

BACKGROUND When used as a pretreatment, hydrogen sulfide (H2S) either attenuated or aggravated lung injury. Therefore, we tested the hypothesis whether posttreatment intravenous Na2S (sulfide) may attenuate lung injury. METHODS After blast wave blunt chest trauma or sham procedure, anesthetized and instrumented mice received continuous intravenous sulfide or vehicle while being kept at 37°C or 32°C core temperature. After 4 hours of pressure-controlled, thoracopulmonary compliance-titrated, lung-protective mechanical ventilation, blood and tissue were harvested for cytokine concentrations, heme oxygenase-1, IκBα, Bcl-Xl, and pBad expression (western blotting), nuclear factor-κB activation (electrophoretic mobility shift assay), and activated caspase-3, cystathionine-β synthase and cystathionine-γ lyase (immunohistochemistry). RESULTS Hypothermia caused marked bradycardia and metabolic acidosis unaltered by sulfide. Chest trauma impaired thoracopulmonary compliance and arterial Po2, again without sulfide effect. Cytokine levels showed inconsistent response. Sulfide increased nuclear factor-κB activation during normothermia, but this effect was blunted during hypothermia. While histologic lung injury was variable, both sulfide and hypothermia attenuated the trauma-related increase in heme oxygenase-1 expression and activated caspase-3 staining, which coincided with increased Bad phosphorylation and Bcl-Xl expression. Sulfide and hypothermia also attenuated the trauma-induced cystathionine-β synthase and cystathionine-γ lyase expression. CONCLUSIONS Posttreatment sulfide infusion after blunt chest trauma did not affect the impaired lung mechanics and gas exchange but attenuated stress protein expression and apoptotic cell death. This protective effect was amplified by moderate hypothermia. The simultaneous reduction in cystathionine-β synthase and cystathionine-γ lyase expression supports the role of H2S-generating enzymes as an adaptive response during stress states.

Effects of intravenous sulfide during resuscitated porcine hemorrhagic shock

Objective:Controversial data are available on the effects of hydrogen sulfide during hemorrhage. Because the clinical significance of hydrogen sulfide administration in rodents may not be applicable to larger species, we tested the hypothesis whether intravenous Na2S (sulfide) would beneficially influence organ dysfunction during long-term, porcine hemorrhage and resuscitation. Design:Prospective, controlled, randomized study. Setting:University animal research laboratory. Subjects:Forty-five domestic pigs of either gender. Interventions:Anesthetized and instrumented animals underwent 4 hrs of hemorrhage (removal of 40% of the blood volume and subsequent blood removal/retransfusion to maintain mean arterial pressure at 30 mm Hg). Sulfide infusion was started 2 hrs before hemorrhage, simultaneously with blood removal or at the beginning of retransfusion of shed blood, and continued for 12 hrs. Resuscitation comprised hydroxyethyl starch and norepinenephrine infusion titrated to maintain mean arterial pressure at preshock values. Measurements and Main Results:Before, immediately at the end of and 12 and 22 hrs after hemorrhage, we measured systemic and regional hemodynamics (portal vein, hepatic and right kidney artery ultrasound flow probes) and oxygen transport, nitric oxide and cytokine production (nitrate+nitrite, interleukin-6, tumor necrosis factor-&agr; levels). Postmortem biopsies were analyzed for histomorphology (hematoxylin and eosin staining) and DNA damage (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling staining). The progressive kidney (creatinine levels, creatinine clearance), liver (transaminase activities, bilirubin levels), and cardiocirculatory (norepipnehrine requirements, troponin I levels) dysfunction was attenuated in the simultaneous treatment group only, which coincided with reduced lung, liver, and kidney histological damage. Sulfide reduced mortality, however, irrespective of the timing of its administration. Conclusions:While the sulfide-induced protection against organ injury was only present when initiated simultaneously with blood removal, it was largely unrelated to hypothermia. The absence of sulfide-mediated protection in the pretreatment protocol may be due to the accumulation of sulfide during low flow states. In conclusion, sulfide treatment can be effective in hemorrhagic shock, but its effectiveness is restricted to a narrow timing and dosing window.

Temperature and cell-type dependency of sulfide effects on mitochondrial respiration.

ABSTRACT Previous studies suggest that sulfide-induced inhibition of cytochrome c oxidase (cCox) and, consequently, the metabolic and toxic effects of sulfide are less pronounced at low body temperature. Because the temperature-dependent effects of sulfide on the inflammatory response are still a matter of debate, we investigated the impact of varying temperature on the cCox excess capacity and the mitochondrial sulfide oxidation by the sulfide-ubiquinone oxidoreductase in macrophage-derived cell lines (AMJ2-C11 and RAW 264.7). Using an oxygraph chamber, the inhibition of mitochondrial respiration was measured by stepwise titrations with sulfide and the nonmetabolizable cCox inhibitor sodium azide at 25°C and 37°C. Using the latter of the two inhibitors, the excess capacity of the cCox was obtained. Furthermore, we quantified the capacity of these cells to withstand sulfide inhibition by measuring the amount required to inhibit respiration by 50% and 90% and the viability of the cells after 24-h exposure to 100 ppm of hydrogen sulfide. At low titration rates, the AMJ2-C11 cells, but not the RAW 264.7 cells, increased their capacity to withstand exogenously added sulfide. This effect was even greater at 25°C than at 37°C. Furthermore, only the AMJ2-C11 cells remained viable after sulfide exposure for 24 h. In contrast, only in the RAW 264.7 cells that an increase in cCox excess capacity was found at low temperatures. In macrophage-derived cell lines, both the excess capacity of cCox and the efficiency of sulfide elimination may increase at low temperatures. These properties may modify the effects of sulfide in immune cells and, potentially, the inflammatory response during sulfide exposure at different body temperatures.

Adrenomedullin binding improves catecholamine responsiveness and kidney function in resuscitated murine septic shock

PurposeAdrenomedullin (ADM) has been referred to as a double-edged sword during septic shock: On one hand, ADM supplementation improved organ perfusion and function, attenuated systemic inflammation, and ultimately reduced tissue apoptosis and mortality. On the other hand, ADM overproduction can cause circulatory collapse and organ failure due to impaired vasoconstrictor response and reduced myocardial contractility. Since most of these data originate from un-resuscitated shock models, we tested the hypothesis whether the newly developed anti-ADM antibody HAM1101 may improve catecholamine responsiveness and thus attenuate organ dysfunction during resuscitated murine, cecal ligation and puncture (CLP)-induced septic shock.MethodsImmediately after CLP, mice randomly received vehicle (phosphate-buffered saline, n = 11) or HAM1101 (n = 9; 2 μg·g−1). Fifteen hours after CLP, animals were anesthetized, mechanically ventilated, instrumented, and resuscitated with hydroxyethylstarch and continuous i.v. norepinephrine to achieve normotensive hemodynamics (mean arterial pressure > 50 to 60 mmHg).ResultsHAM1101 pretreatment reduced the norepinephrine infusion rates required to achieve hemodynamic targets, increased urine flow, improved creatinine clearance, and lowered neutrophil gelatinase-associated lipocalin blood levels, which coincided with reduced expression of the inducible nitric oxide synthase and formation of peroxynitrite (nitrotyrosine immunostaining) in the kidney and aorta, ultimately resulting in attenuated systemic inflammation and tissue apoptosis.ConclusionsDuring resuscitated murine septic shock, early ADM binding with HAM1101 improved catecholamine responsiveness, blunted the shock-related impairment of energy metabolism, reduced nitrosative stress, and attenuated systemic inflammatory response, which was ultimately associated with reduced kidney dysfunction and organ injury.

Effects of Hyperoxia and Mild Therapeutic Hypothermia During Resuscitation From Porcine Hemorrhagic Shock.

Objective:Hemorrhagic shock–induced tissue hypoxia induces hyperinflammation, ultimately causing multiple organ failure. Hyperoxia and hypothermia can attenuate tissue hypoxia due to increased oxygen supply and decreased demand, respectively. Therefore, we tested the hypothesis whether mild therapeutic hypothermia and hyperoxia would attenuate postshock hyperinflammation and thereby organ dysfunction. Design:Prospective, controlled, randomized study. Setting:University animal research laboratory. Subjects:Thirty-six Bretoncelles-Meishan-Willebrand pigs of either gender. Interventions:After 4 hours of hemorrhagic shock (removal of 30% of the blood volume, subsequent titration of mean arterial pressure at 35 mm Hg), anesthetized and instrumented pigs were randomly assigned to “control” (standard resuscitation: retransfusion of shed blood, fluid resuscitation, norepinephrine titrated to maintain mean arterial pressure at preshock values, mechanical ventilation titrated to maintain arterial oxygen saturation > 90%), “hyperoxia” (standard resuscitation, but FIO2, 1.0), “hypothermia” (standard resuscitation, but core temperature 34°C), or “combi” (hyperoxia plus hypothermia) (n = 9 each). Measurements and Main Results:Before, immediately at the end of and 12 and 22 hours after hemorrhagic shock, we measured hemodynamics, blood gases, acid-base status, metabolism, organ function, cytokine production, and coagulation. Postmortem kidney specimen were taken for histological evaluation, immunohistochemistry (nitrotyrosine, cystathionine &ggr;-lyase, activated caspase-3, and extravascular albumin), and immunoblotting (nuclear factor-&kgr;B, hypoxia-inducible factor-1&agr;, heme oxygenase-1, inducible nitric oxide synthase, B-cell lymphoma-extra large, and protein expression of the endogenous nuclear factor-&kgr;B inhibitor). Although hyperoxia alone attenuated the postshock hyperinflammation and thereby tended to improve visceral organ function, hypothermia and combi treatment had no beneficial effect. Conclusions:During resuscitation from near-lethal hemorrhagic shock, hyperoxia attenuated hyperinflammation, and thereby showed a favorable trend toward improved organ function. The lacking efficacy of hypothermia was most likely due to more pronounced barrier dysfunction with vascular leakage–induced circulatory failure.

Effects of pretreatment hypothermia during resuscitated porcine hemorrhagic shock.

Objectives:Accidental hypothermia increases mortality and morbidity after hemorrhage, but controversial data are available on the effects of therapeutic hypothermia. Therefore, we tested the hypothesis whether moderate pretreatment hypothermia would beneficially influence organ dysfunction during long-term, porcine hemorrhage and resuscitation. Design:Prospective, controlled, randomized study. Setting:University animal research laboratory. Subjects:Twenty domestic pigs of either gender. Interventions:Using an extracorporeal heat exchanger, anesthetized and instrumented animals were maintained at 38°C, 35°C, or 32°C core temperature and underwent 4 hours of hemorrhage (removal of 40% of the blood volume and subsequent blood removal/retransfusion to maintain mean arterial pressure at 30 mm Hg). Resuscitation comprised of hydroxyethyl starch and norepinephrine infusion titrated to maintain mean arterial pressure at preshock values. Measurements and Main Results:Before, immediately at the end of, and 12 and 22 hours after hemorrhage, we measured systemic and regional hemodynamics (portal vein, hepatic and right kidney artery ultrasound flow probes) and oxygen transport, and nitric oxide and cytokine production. Hemostasis was assessed by rotation thromboelastometry. Postmortem biopsies were analyzed for histomorphology (hematoxylin and eosin staining) and markers of apoptosis (kidney Bcl-xL and caspase-3 expression). Hypothermia at 32°C attenuated the shock-related lactic acidosis but caused metabolic acidosis, most likely resulting from reduced carbohydrate oxidation. Although hypothermia did not further aggravate shock-related coagulopathy, it caused a transitory attenuation of kidney and liver dysfunction, which was ultimately associated with reduced histological damage and more pronounced apoptosis. Conclusions:During long-term porcine hemorrhage and resuscitation, moderate pretreatment hypothermia was associated with a transitory attenuation of organ dysfunction and less severe histological tissue damage despite more pronounced metabolic acidosis. This effect is possibly due to a switch from necrotic to apoptotic cell death, ultimately resulting from reduced tissue energy deprivation during the shock phase.

Exposure to 100% Oxygen Abolishes the Impairment of Fracture Healing after Thoracic Trauma

In polytrauma patients a thoracic trauma is one of the most critical injuries and an important trigger of post-traumatic inflammation. About 50% of patients with thoracic trauma are additionally affected by bone fractures. The risk for fracture malunion is considerably increased in such patients, the pathomechanisms being poorly understood. Thoracic trauma causes regional alveolar hypoxia and, subsequently, hypoxemia, which in turn triggers local and systemic inflammation. Therefore, we aimed to unravel the role of oxygen in impaired bone regeneration after thoracic trauma. We hypothesized that short-term breathing of 100% oxygen in the early post-traumatic phase ameliorates inflammation and improves bone regeneration. Mice underwent a femur osteotomy alone or combined with blunt chest trauma 100% oxygen was administered immediately after trauma for two separate 3 hour intervals. Arterial blood gas tensions, microcirculatory perfusion and oxygenation were assessed at 3, 9 and 24 hours after injury. Inflammatory cytokines and markers of oxidative/nitrosative stress were measured in plasma, lung and fracture hematoma. Bone healing was assessed on day 7, 14 and 21. Thoracic trauma induced pulmonary and systemic inflammation and impaired bone healing. Short-term exposure to 100% oxygen in the acute post-traumatic phase significantly attenuated systemic and local inflammatory responses and improved fracture healing without provoking toxic side effects, suggesting that hyperoxia could induce anti-inflammatory and pro-regenerative effects after severe injury. These results suggest that breathing of 100% oxygen in the acute post-traumatic phase might reduce the risk of poorly healing fractures in severely injured patients.

Association of Kidney Tissue Barrier Disrupture and Renal Dysfunction in Resuscitated Murine Septic Shock.

Abstract Septic shock-related kidney failure is characterized by almost normal morphological appearance upon pathological examination. Endothelial barrier disrupture has been suggested to be of crucial importance for septic shock-induced organ dysfunction. Therefore, in murine resuscitated cecal ligation and puncture (CLP)-induced septic shock, we tested the hypothesis whether there is a direct relationship between the kidney endothelial barrier injury and renal dysfunction. Anesthetized mice underwent CLP, and 15 h later, were anesthetized again and surgically instrumented for a 5-h period of intensive care comprising lung-protective mechanical ventilation, fluid resuscitation, continuous i.v. norepinephrine to maintain target hemodynamics, and measurement of creatinine clearance (CrCl). Animals were stratified according to low or high CrCl. Nitrotyrosine formation, expression of the inducible isoform of the nitric oxide synthase, and blood cytokine (tumor necrosis factor, interleukin-6, interleukin-10) and chemokine (monocyte chemoattractant protein-1, keratinocyte-derived chemokine) levels were significantly higher in animals with low CrCl. When plotted against CrCl and neutrophil gelatinase-associated lipocalin levels, extravascular albumin accumulation, and tissue expression of the vascular endothelial growth factor and angiopoietin-1 showed significant mathematical relationships related to kidney (dys)function. Preservation of the constitutive expression of the hydrogen sulfide producing enzyme cystathione-&ggr;-lyase was associated with maintenance of organ function. The direct quantitative relation between microvascular leakage and kidney (dys)function may provide a missing link between near-normal tissue morphology and septic shock-related renal failure, thus further highlighting the important role of vascular integrity in septic shock-related renal failure.

Physiological and immune-biological characterization of a long-term murine model of blunt chest trauma.

ABSTRACT Blunt chest trauma causes pulmonary and systemic inflammation. It is still a matter of debate whether the long-term course of this inflammatory response is associated with persistent impairment of lung function. We hypothesized that an increase of inflammatory biomarkers may still be present at later time points after blunt chest trauma, eventually, despite normalized lung mechanics and gas exchange. Anesthetized spontaneously breathing male C57BL/6J mice underwent a blast wave–induced blunt chest trauma or sham procedure. Twelve and 24 h later, blood gases and lung mechanics were measured, together with blood, bronchoalveolar lavage (BAL), and tissue cytokine concentrations (multiplex cytokine kit); heme oxygenase 1 (HO-1), activated caspase-3, Bcl-xL, and Bax expression (Western blotting); nuclear factor-&kgr;B activation (electrophoretic mobility shift assay); nitrotyrosine formation; and purinergic (P2XR4 and P2XR7) receptor expression (immunohistochemistry). Histological damage was assessed by hematoxylin and eosin and periodic acid-Schiff staining. High-resolution respirometry allowed assessing mitochondrial respiration in diaphragm biopsies. Chest trauma significantly increased tissue and BAL cytokine levels, associated with a significant increase in HO-1, purinergic receptor expression, and tissue nitrotyrosine formation. In contrast, lung mechanics, gas exchange, and histological damage did not show any significant difference between sham and trauma groups. Activation of the immune response remains present at later time points after murine blunt chest trauma. Discordance of the increased local inflammatory response and preserved pulmonary function may be explained by a dissociation of the immune response and lung function, such as previously suggested after experimental sepsis.

Blunt Chest Trauma in Mice after Cigarette Smoke-Exposure: Effects of Mechanical Ventilation with 100 % O2

Cigarette smoking (CS) aggravates post-traumatic acute lung injury and increases ventilator-induced lung injury due to more severe tissue inflammation and apoptosis. Hyper-inflammation after chest trauma is due to the physical damage, the drop in alveolar PO2, and the consecutive hypoxemia and tissue hypoxia. Therefore, we tested the hypotheses that 1) CS exposure prior to blunt chest trauma causes more severe post-traumatic inflammation and thereby aggravates lung injury, and that 2) hyperoxia may attenuate this effect. Immediately after blast wave-induced blunt chest trauma, mice (n=32) with or without 3-4 weeks of CS exposure underwent 4 hours of pressure-controlled, thoraco-pulmonary compliance-titrated, lung-protective mechanical ventilation with air or 100 % O2. Hemodynamics, lung mechanics, gas exchange, and acid-base status were measured together with blood and tissue cytokine and chemokine concentrations, heme oxygenase-1 (HO-1), activated caspase-3, and hypoxia-inducible factor 1-α (HIF-1α) expression, nuclear factor-κB (NF-κB) activation, nitrotyrosine formation, purinergic receptor 2X4 (P2XR4) and 2X7 (P2XR7) expression, and histological scoring. CS exposure prior to chest trauma lead to higher pulmonary compliance and lower PaO2 and Horovitz-index, associated with increased tissue IL-18 and blood MCP-1 concentrations, a 2-4-fold higher inflammatory cell infiltration, and more pronounced alveolar membrane thickening. This effect coincided with increased activated caspase-3, nitrotyrosine, P2XR4, and P2XR7 expression, NF-κB activation, and reduced HIF-1α expression. Hyperoxia did not further affect lung mechanics, gas exchange, pulmonary and systemic cytokine and chemokine concentrations, or histological scoring, except for some patchy alveolar edema in CS exposed mice. However, hyperoxia attenuated tissue HIF-1α, nitrotyrosine, P2XR7, and P2XR4 expression, while it increased HO-1 formation in CS exposed mice. Overall, CS exposure aggravated post-traumatic inflammation, nitrosative stress and thereby organ dysfunction and injury; short-term, lung-protective, hyperoxic mechanical ventilation have no major beneficial effect despite attenuation of nitrosative stress, possibly due to compensation of by regional alveolar hypoxia and/or consecutive hypoxemia, resulting in down-regulation of HIF-1α expression.