Oscar McCook

Universität Ulm, Ulm, Baden-Württemberg, Germany

Are you Oscar McCook?

Claim your profile

Publications (36)134.84 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: 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 γ-lyase, activated caspase-3, and extravascular albumin), and immunoblotting (nuclear factor-κB, hypoxia-inducible factor-1α, heme oxygenase-1, inducible nitric oxide synthase, B-cell lymphoma-extra large, and protein expression of the endogenous nuclear factor-κB 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.
    No preview · Article · Nov 2015 · Critical care medicine
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Severe tissue trauma-induced systemic inflammation is often accompanied by evident or occult blood-organ barrier dysfunctions, frequently leading to multiple organ dysfunction. However, it is unknown whether specific barrier molecules are shed into the circulation early after trauma as potential indicators of an initial barrier dysfunction. The release of the barrier molecule junctional adhesion molecule-1 (JAM-1) was investigated in plasma of C57BL/6 mice 2 h after experimental mono- and polytrauma as well as in polytrauma patients (ISS ≥ 18) during a 10-day period. Correlation analyses were performed to indicate a linkage between JAM-1 plasma concentrations and organ failure. JAM-1 was systemically detected after experimental trauma in mice with blunt chest trauma as a driving force. Accordingly, JAM-1 was reduced in lung tissue after pulmonary contusion and JAM-1 plasma levels significantly correlated with increased protein levels in the bronchoalveolar lavage as a sign for alveolocapillary barrier dysfunction. Furthermore, JAM-1 was markedly released into the plasma of polytrauma patients as early as 4 h after the trauma insult and significantly correlated with severity of disease and organ dysfunction (APACHE II and SOFA score). The data support an early injury- and time-dependent appearance of the barrier molecule JAM-1 in the circulation indicative of a commencing trauma-induced barrier dysfunction.
    Full-text · Article · Nov 2015 · Mediators of Inflammation
  • [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: Acute and chronic lung disease up-regulate purinergic receptor (P2XR4, P2XR7) expression. P2XR7 deletion or blockade attenuated pulmonary hyper-inflammation, but P2XR4 up-regulation compensated for P2XR7 deletion. Therefore, we tested the hypotheses that P2XR4 deletion may attenuate post-traumatic acute lung injury (ALI) after cigarette smoke (CS) exposure-induced COPD. Methods: After 3-4 weeks of CS exposure, anesthetized wild type or P2XR4-ko mice (n = 8 each) underwent pressure wave-induced blunt chest trauma followed by 4 hours of lung-protective mechanical ventilation, fluid resuscitation and noradrenaline to maintain blood pressure > 55mmHg. Lung mechanics, gas exchange, hemodynamics, metabolism and acid-base status were measured together with lung histology,immune-histochemistry, and western blotting. Results: P2XR4-ko mice showed higher lung compliance and lower PaO2/FiO2 ratios, which coincided with higher P2XR7 expression, aggravated histological damage and immune-cell infiltration and HO-1 expression. In contrast, P2XR4 deletion was associated with less impairment of systemic hemodynamics, glucose homeostasis and acid-base status. Conclusion: After CS exposure, genetic P2XR4 deletion aggravated post-traumatic ALI and hyper-inflammation, but attenuated impairment of systemic hemodynamics and metabolism, possibly due to preserved liver metabolic capacity resulting from less alveolar hypoxia-induced right ventricular re-modelling. Acknowledgements: Supported by Ministry of Science, Research and the Arts of Baden-Württemberg (Az:32-729.55-0/239-5/32-7533.-6-10/15/1) (Boehringer Ingelheim Ulm University BioCenter).
    No preview · Article · Sep 2015 · Shock (Augusta, Ga.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: Hemorrhagic shock (HS) causes tissue hypoxia, increased oxidative stress and hyper-inflammation. Therapeutic hypothermia may decrease mortality through reduced inflammation and hypometabolism, but may be associated with loss of plasma, potentially compromising circulation. Therefore, we investigated the effects of moderate therapeutic hypothermia on immune and barrier dysfunction (BD) in a long-term model of resuscitated HS. Methods: After volume- and pressure controlled HS of 3 hours (withdrawal of 30% of calculated blood volume and titration to mean arterial pressure (MAP) ∼40 mmHg), anaesthetized, mechanically ventilated, and instrumented pigs were randomized to either normothermia (38°C) or hypothermia (34°C over 12 hrs → rewarmed) (n = 9 each) during a total of 23 hours of resuscitation (re-transfusion of shed blood, fluids, and noradrenaline to maintain MAP). Parameters of organ function and inflammation were measured before and at the end of HS, and at 12 and 23 hours of resuscitation. Paraffin-embedded postmortem kidney biopsies were analyzed for markers of BD (albumin extravasation) and oxidative/nitrosative stress with immune-histochemistry. Results: Hypothermic animals needed significantly higher noradrenaline doses (p = 0.013) to maintain MAP, and showed higher hemoglobin concentrations (p = 0.003) during cooling. Hypothermia did not improve organ dysfunction. Conclusion: Therapeutic hypothermia showed no benefit, most likely as a result of fluid shift into the extra-vascular space. Acknowledgement: Supported by the German Department of Defense (AZ E/U2AD/CF523/DF556).
    No preview · Article · Sep 2015 · Shock (Augusta, Ga.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: Hemorrhagic shock-induced tissue hypoxia induces hyper-inflammation, ultimately causing multiple organ failure. Hyperoxia and hypothermia can attenuate tissue hypoxia due to increased O2 supply and decreased demand, respectively. Therefore, we tested the hypothesis whether mild therapeutic hypothermia and hyperoxia would attenuate post-shock hyper-inflammation and thereby organ dysfunction. Methods: After four hours of hemorrhage (removal of 30% of the blood volume, subsequent titration of mean arterial pressure (MAP) at 35 mmHg), anesthetized and instrumented pigs were randomly assigned to "control" (standard resuscitation: re-transfusion of shed blood, fluid resuscitation, norepinephrine titrated to maintain MAP at pre-shock values, mechanical ventilation titrated to maintain arterial O2 saturation > 90%), "hyperoxia" (standard resuscitation, but inspired O2 fraction (FiO2) 1.0), "hypothermia" (standard resuscitation, but core temperature 34°C), or "combi" (hyperoxia plus hypothermia) (n = 9 each). Before, immediately at the end of, and 12 and 22 hours after hemorrhage, we measured hemodynamics, blood gases, acid-base status, metabolism, organ function, cytokine production, and coagulation. Post-mortem kidney biopsies were taken for histological evaluation, immuno-histochemistry (nitrotyrosine, cystathionine gamma-lyase, activated caspase-3, and extravascular albumine), and immuno-blotting (NF-kB, HIF-1 alpha, HO-1, iNOS, Bcl-xL, and IkB alpha). Results: While hyperoxia alone attenuated the post-shock hyper-inflammation and thereby visceral organ dysfunction, hypothermia and "combi" treatment had no beneficial effect. Conclusions: During resuscitation from near-lethal hemorrhagic shock, hyperoxia attenuated hyper-inflammation and thereby organ dysfunction. The lacking efficacy of hypothermia was most likely due to aggravated barrier dysfunction with vascular leakage-induced circulatory failure. Acknowledgement: Supported by the Bundesministerium der Verteidigung (Vertragsforschungsvorhaben AZ E/U2AD/CF523/DF556).
    No preview · Article · Sep 2015 · Shock (Augusta, Ga.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: Co-morbidities are a confounding factor increasing intensive care mortality. Atherosclerosis and chronic kidney disease (CKD) not only influence renal function, e.g. reduce glomerular filtration rate (GFR), but may also cause endothelial barrier dysfunction (BD). Fluid extravasation due to BD is a risk factor in circulatory shock. In murine CKD models, ambivalent data are reported on regulating mechanisms of BD. Therefore, we investigated the role of BD in swine with reduced GFR, atherosclerosis and impaired tissue receptor protein expression (EPO-R, PPAR-β). Methods: On paraffin-embedded kidney biopsies taken during surgical instrumentation of young and healthy German-Landrace (GLR) and FBM (Familial Hypercholesterolemia, Bretoncelles, Meishian) pigs fed with a high fat diet (n = 8 each) immune-histochemistry was performed to detect extravascular albumin accumulation (Alb), and tissue Angiopoietin 1 (Ang 1), vascular endothelial cadherin (VE-Cad), and vascular endothelial growth factor (VEGF) expression. Results: Co-morbidity was associated with significantly higher Alb-extravasation (p = 0.007), VEGF (p = 0.03) and VE-Cad (p = 0.017). Elevated Alb- and VEGF-levels indicate increased BD. Increased VE-Cad expression, an adherens protein regulating and stabilizing endothelial function, which contrasts with the otherwise VEGF-induced VE-Cad degradation during BD, corroborates data on murine ureter obstruction-induced CKD. Conclusion: Co-morbidity leads to BD as shown by increased Alb extravasation; further studies are needed to investigate this altered mechanism of BD in co-morbidity models. Acknowledgement: Supported by the DFG (SFB 1149).
    No preview · Article · Sep 2015 · Shock (Augusta, Ga.)
  • F Zink · T Stenzel · U Wachter · J Vogt · O McCook · P Radermacher
    [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: Septic shock causes diffuse microvascular leakage leading to interstitial edema, and hypovolemia. In a resuscitated murine CLP-induced septic shock model that the adrenomedullin antibody HAM1101 reduced norepinephrine requirements, increased urine flow, ameliorated kidney dysfunction and organ injury. To dissect the mechanisms by which endothelial barrier injury may contribute to kidney dysfunction we plotted creatinine clearance as a function of bio-markers for barrier dysfunction. Methods: 15 hours after CLP and random assignment to vehicle or HAM1101, anesthetized, mechanically ventilated, and instrumented mice underwent 5 hours of lung-protective mechanical ventilation, fluid resuscitation and continuous i.v. norepinephrine to maintain target hemodynamics. We analyzed post-hoc paraffin sections from this previous study by immunohistochemistry for kidney tissue expression of albumin, vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang1), and the H2S-producing enzymes cystathionine-g-lyase (CSE) and -b-synthase (CBS). For each individual experimental animal values were plotted against the creatinine clearance. Results: Extra-vascular albumin accumulation (r = -0.71) and the signaling molecules associated with maintaining endothelial vascular integrity were significantly related with kidney function (Ang-1: r = 0.63; VEGF: r = -0.69). Furthermore, we could confirm results associating preservation of constitutive CBS (r = 0.69) and CSE (r = 0.64) expression with improved kidney function. Conclusion: We were able to demonstrate a direct link between microvascular leakage and kidney function, further highlighting the important role of vascular integrity in septic shock-related renal failure.
    No preview · Article · Sep 2015 · Shock (Augusta, Ga.)
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Full-text · Article · Aug 2015 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Full-text · Article · Jul 2015 · PLoS ONE

  • No preview · Article · Apr 2015 · Shock
  • [Show abstract] [Hide abstract]
    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. 12 and 24 hours 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-κB activation (electrophoretic mobility shift assay), nitrotyrosine formation, and purinergic (P2XR4 and P2XR7) receptor expression (immunohistochemistry). Histological damage was assessed by HE and PAS 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 of 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.
    No preview · Article · Feb 2015 · Shock
  • [Show abstract] [Hide abstract]
    ABSTRACT: Numerous papers have been published on the role of H2S during circulatory shock. Consequently, knowledge about vascular sulfide concentrations may assume major importance, in particular in the context of “acute on chronic disease”, i.e., during circulatory shock in animals with pre-existing chronic disease. This review addresses the questions i) of the “real” sulfide levels during circulatory shock, and, ii) to which extent injury and pre-existing co-morbidity may affect the expression of H2S producing enzymes under these conditions. In the literature there is a huge range on sulfide blood levels during circulatory shock, in part as a result of the different analytical methods used, but also due to the variable of the models and species studied. Clearly, some of the very high levels reported should be questioned in the context of the well-known H2S toxicity. As long as “real” sulfide levels during circulatory shock are unknown and/or undetectable “on line” due to the lack of appropriate techniques, it appears to be premature to correlate the measured blood levels of hydrogen sulfide with the severity of shock or the H2S therapy-related biological outcomes. The available data on the tissue expression of the H2S-releasing enzymes during circulatory shock suggest that a “constitutive” CSE expression may play a crucial role of for the maintenance of organ function, at least in the kidney. The data also indicate that increased CBS and CSE expression, in particular in the lung and the liver, represents an adaptive response to stress states.
    No preview · Article · Sep 2014 · Nitric Oxide
  • [Show abstract] [Hide abstract]
    ABSTRACT: Our aim was to study the ability of an immortalized cell line (AMJ2-C11) to sustain aerobic cell respiration at decreasing oxygen concentrations under continuous sulfide exposure. We assumed that the rate of elimination of sulfide through the pathway linked to the mitochondrial respiratory chain and therefore operating under aerobic conditions, should decrease with limiting oxygen concentrations. Thus, sulfide's inhibition of cellular respiration would occur faster under continuous sulfide exposure when the oxygen concentration is in the very low range. The experiments were performed with an O2K-oxygraph (Oroboros Instruments) by suspending 0.5 - 1 x 10(6) cells in 2 ml of continuously stirred respiration medium at 37°C and calculating the oxygen flux (JO2) as the negative derivative of the oxygen concentration in the medium. The cells were studied in two different metabolic states, namely under normal physiologic respiration (1) and after uncoupling of mitochondrial respiration (2). Oxygen concentration was controlled by means of a titration-injection pump, resulting in average concentration values of 0.73 ± 0.05 μM, 3.1 ± 0.2 μM, and 6.2 ± 0.2 μM. Simultaneously we injected a 2 mM Na2S solution at a continuous rate of 10 μl/s in order to quantify the titration-time required to reduce the JO2 to 50% of the initial respiratory activity. Under the lowest oxygen concentration this effect was achieved after 3.5 [0.3;3.5] and 11.7 [6.2;21.2] min in the uncoupled and coupled state, respectively. This time was statistically significantly shorter when compared to the intermediate and the highest O2 concentrations tested, which yielded values of 24.6[15.5;28.1] min (coupled) and 35.9[27.4;59.2] min (uncoupled), as well as 42.4 [27.5;42.4] min (coupled) and 51.5 [46.4;51.7] min (uncoupled). All data are medians [25%, and 75% percentiles]. Our results confirm that the onset of inhibition of cell respiration by sulfide occurs earlier under a continuous exposure when approaching the anoxic condition. This property may contribute to the physiological role of sulfide as an oxygen sensor.
    No preview · Article · Jun 2014 · Nitric Oxide
  • O. McCook · F. Wagner · A. Scheuerle · M. Groeger · A. Bergmann · F. Hein · J. Struck · P. Radermacher · K. Wagner

    No preview · Conference Paper · Jun 2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Our aim was to study the ability of an immortalized cell line (AMJ2-C11) to sustain aerobic cell respiration at decreasing oxygen concentrations under continuous sulfide exposure. We assumed that the rate of elimination of sulfide through the pathway linked to the mitochondrial respiratory chain and therefore operating under aerobic conditions, should decrease with limiting oxygen concentrations. Thus, sulfide’s inhibition of cellular respiration would occur faster under continuous sulfide exposure when the oxygen concentration is in the very low range. The experiments were performed with an O2K-oxygraph (Oroboros Instruments) by suspending 0.5–1 × 106 cells in 2 ml of continuously stirred respiration medium at 37 °C and calculating the oxygen flux (JO2) as the negative derivative of the oxygen concentration in the medium. The cells were studied in two different metabolic states, namely under normal physiologic respiration (1) and after uncoupling of mitochondrial respiration (2). Oxygen concentration was controlled by means of a titration-injection pump, resulting in average concentration values of 0.73 ± 0.05 μM, 3.1 ± 0.2 μM, and 6.2 ± 0.2 μM. Simultaneously we injected a 2 mM Na2S solution at a continuous rate of 10 μl/s in order to quantify the titration-time required to reduce the JO2 to 50% of the initial respiratory activity. Under the lowest oxygen concentration this effect was achieved after 3.5 [0.3;3.5] and 11.7 [6.2;21.2] min in the uncoupled and coupled state, respectively. This time was statistically significantly shorter when compared to the intermediate and the highest O2 concentrations tested, which yielded values of 24.6 [15.5;28.1] min (coupled) and 35.9 [27.4;59.2] min (uncoupled), as well as 42.4 [27.5;42.4] min (coupled) and 51.5 [46.4;51.7] min (uncoupled). All data are medians [25%, and 75% percentiles]. Our results confirm that the onset of inhibition of cell respiration by sulfide occurs earlier under a continuous exposure when approaching the anoxic condition. This property may contribute to the physiological role of sulfide as an oxygen sensor.
    No preview · Article · Jan 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Adrenomedullin (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. Immediately 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). HAM1101 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. During 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.
    Full-text · Article · Oct 2013
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: In un-resuscitated rodent models of septic shock, the peroxisome proliferator-activated receptor-beta/delta (PPAR-beta/delta) agonist GW0742 improved visceral organ function. Therefore, we tested the hypothesis whether GW0742 would attenuate kidney injury during long-term, resuscitated, porcine polymicrobial septic shock. Methods: Six, 12, and 18h after the induction of fecal peritonitis by inoculation of autologous feces, anesthetized, mechanically ventilated, and instrumented male pigs with pre-existing atherosclerosis resulting from familial hypercholesteremia and atherogenic diet randomly received either vehicle (dimethyl sulfoxide, n = 12) or GW0742 (n = 10). Resuscitation comprised hydroxyethyl starch and norepinephrine infusion titrated to maintain mean arterial pressure at baseline values. Results: Despite aggressive fluid resuscitation, fecal peritonitis was associated with arterial hypotension requiring norepinephrine infusion, ultimately resulting in progressive lactic acidosis and acute kidney injury. GW0742 did not beneficially affect any parameter of systemic and regional hemodynamics, gas exchange, metabolism, or organ function. The parameters of inflammation, oxidative and nitrosative stress, and organ injury (post-mortem analysis for histomorphology and markers of apoptosis) were not influenced either. Immunohistochemistry of pre-shock kidney biopsies from a previous study in this swine strain showed markedly lower PPAR-beta/delta receptor expression than in healthy animals. Conclusions: In swine with pre-existing atherosclerosis, the PPAR-beta/delta agonist GW0742 failed to attenuate septic shock-induced circulatory failure and kidney dysfunction, most likely due to reduced receptor expression coinciding with cardiovascular and metabolic co-morbidity.
    Full-text · Article · Oct 2013
  • [Show abstract] [Hide abstract]
    ABSTRACT: H2S production in the kidney, both before and after ischemic injury, and its regulation via endogenous synthesizing enzymes: cystathionine β synthase (CBS), γ lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST) remains controversial. The conflicting results were all reported in non-resuscitated young and healthy rodent models that have come into question with regard to their applicability to humans, let alone the co-morbidity frequently present in ICU patients. To understand the endogenous H2S synthesis in disease states, we compared kidneys from swine strains with and without pre-existing cardiovascular co-morbidity. CSE, CBS and MST expression was quantified by immunohistochemistry (densitometric image analysis, presented as mean sum of densitometric units x10(5)) of formalin-fixed paraffin sections from both pre-injury and post-ischemia/reperfusion (I/R) kidney biopsies taken in young and healthy pigs (German Landrace, n=8 ) as well as swine strain with familial hypercholesterolemia (FBM, n=8) (11.1 (7.4;12.3) vs. 1.4 (1.3;1.5) mmol/L, p<0.01, n=20 and 15, resp.) and consecutive, diet-induced atherosclerosis. Arterial blood 8-isoprostane (commercially available ELISA kit) and nitrite+nitrate (chemiluminescence) concentrations were measured as markers of lipid peroxidation and NO production. Urine was sampled during 2 hours before aortic occlusion and 8 hours of reperfusion. Urinary and blood creatinine and Na(+) levels allowed calculating creatinine clearance (glomerular filtration) and fractional Na(+) excretion (tubular re-absorption). Atherosclerotic swine presented with reduced glomerular filtration (creatinine clearance 76 (60;83) vs. 103 (79;120) mL/min, n=19 each, p<0.004) and chronic histological kidney injury (dilatation of Bowman's space, swelling of Bowman's capsule, tubular dilatation). Baseline isoprostane levels were higher than in the healthy German Landrace swine (111±47 vs. 74±16pg/mL, p<0.005), while endogenous NO production rate was lower as indicated by the lower nitrate+nitrite levels (baseline levels 140±360 vs. 770±800μmol/L, p<0.001). There was no difference in the CSE expression of native kidneys specimens (1520 (1430;1570) vs. 1590 (1430;1590) AU). Kidney I/R injury was associated with down-regulation of CSE expression, which was significantly more pronounced in the FBM swine (400 (280;460) AU, p=0.003 vs. before I/R injury; young and healthy German landrace swine 1110 (1020;1210) AU, p=0.028 vs. before I/R injury; p=0.001 between groups). CBS expression was not present in native biopsies, and minimal only post I/R, i.e. in necrotic tubules and cells only. FBM swine presented with higher MST expression prior to I/R injury (p=0.030 vs. healthy German Landrace swine). I/R injury increased MST expression (FBM swine 476 (200;770) vs. 91 (30;150) AU; young and healthy German Landrace swine (156 (138;231) vs. 0.06 (0.001;1) AU; both p=0.004 vs. before I/R), the difference between FBM and healthy domestic swine being less pronounced than prior to kidney I/R (p=0.002 between groups). I/R injury caused a down-regulation of the CSE enzyme in young and healthy swine. This response was significantly more pronounced in swine with underlying atherosclerotic disease, possibly due to systemically reduced NO and increased reactive oxygen species formation. MST up-regulation coincides with increased kidney injury and may be playing a detoxifying role.
    No preview · Article · Sep 2013 · Nitric Oxide
  • [Show abstract] [Hide abstract]
    ABSTRACT: The reported sulfide absorption values and baseline blood concentrations are still controversial [1]. We modified an existing gas chromatographic/mass spectrometric method of routine sulfide quantification using a bis-pentafluorobenzyl derivative [2]. This approach allows sensitive determination of sulfide in small sample volumes under mild chemical conditions. In addition, in in vitro experiments, it allows to distinguish between endogenous and exogenous sulfide by administration of the stable isotope (34)S(2-). A standard protocol utilises 100μl of blood, but can be scaled down to 25μl for rodent samples. In brief, a mixture, consisting of 400μl internal standard (5μg/ml tribromobenzene in isooctane), 200μl of alkylation reagent (10μl/ml pentafluorobenzylbromide in isooctane) and 400μl of phase transfer catalyst (2mg/ml tetradecyldimethylbenzylammonium chloride in sodium tetraborate saturated water) was prepared. After addition of 100μl sample, the mixture was vigorously shaken for 1min, 400μl of saturated potassium dihydrogenphosphate solution were added and the cup was shaken for another 10s. 2μl of the isooctane phase were injected into an Agilent 5890/5970 gas chromatography/mass spectrometry instrument, housing a 12m Macherey-Nagel Optima-5MS capillary column. In sim mode ions m/z 313.7 for the internal standard and 394.0 for the sulfide derivative (or 396.0 for (34)S(2-)) were recorded. The method was used to determine basal sulfide blood concentrations in pigs and to measure the sulfide release from the sulfide releasing drug GYY4137 over 24h in cell culture medium. In addition this method reproducibly was used to determine the absorption of a spike of 100μM (34)S(2-) at pH 7.4 in buffer, blood, plasma, cysteine, glutathione and albumine. Baseline blood sulfide concentrations in pigs were between 0.2 and 2.1μM. Sulfide concentrations of a 300μM solution of GYY4137 in cell culture medium (in cell culture flask, gas exchange enabled) were 1.02μM (10min after GYY addition), 1.32μM (2h), 1.37μM (4h), 1.60μM (8h), 1.58μM (12h) and 1.81μM (24h). The sulfide concentration of the corresponding medium without GYY4137 ranged between 0.25 and 0.50μM. An initial 100μM spike of (34)S(2-) in blood decreased to 23μM after 1min, 12μM after 10 and 1μM after 60min. The corresponding values for plasma were 60, 41 and 3μM, for phosphate buffer (pH 7.4) 103, 96 and 85μM. In a 50mg/ml solution of albumine the (34)S(2-)concentration decreased to 22, 15, 3μM, in 5mg/ml oxidized glutathione to 32, 18, 3μM, in 5mg/ml reduced glutathione to 79, 76, 75μM, in 120μg/ml cystine to 91, 64, 12μM and in 1mg/ml cysteine to 110, 102, 99μM. Our findings show, that sulfide baseline concentrations in blood are in the low micromolar range. Added sulfide is rapidly absorbed by blood, plasma and disulfide bridge containing peptides and proteins, but much lower by the reduced species. Adding the sulfide releasing drug GYY4137, increased the sulfide concentration of cell culture media for 24h.
    No preview · Article · Sep 2013 · Nitric Oxide
  • [Show abstract] [Hide abstract]
    ABSTRACT: The reported sulfide absorption values and baseline blood concentrations are still controversial [1]. We modified an existing gas chromatographic/mass spectrometric method of routine sulfide quantification using a bis-pentafluorobenzyl derivative [2]. This approach allows sensitive determination of sulfide in small sample volumes under mild chemical conditions. In addition, in in vitro experiments, it allows to distinguish between endogenous and exogenous sulfide by administration of the stable isotope (34)S(2-). A standard protocol utilises 100μl of blood, but can be scaled down to 25μl for rodent samples . In brief, a mixture, consisting of 400μl internal standard (5μg/ml tribromobenzene in isooctane), 200μl of alkylation reagent (10μl/ml pentafluorobenzylbromide in isooctane) and 400μl of phase transfer catalyst (2mg/ml tetradecyldimethylbenzylammonium chloride in sodium tetraborate saturated water) was prepared. After addition of 100μl sample, the mixture was vigorously shaken for 1min, 400μl of saturated potassium dihydrogenphosphate solution were added and the cup was shaken for another 10s. 2μl of the isooctane phase were injected into an Agilent 5890/5970 gas chromatography/mass spectrometry instrument, housing a 12m Macherey-Nagel Optima-5MS capillary column. In sim mode ions m/z 313.7 for the internal standard and 394.0 for the sulfide derivative (or 396.0 for (34)S(2-)) were recorded. The method was used to determine basal sulfide blood concentrations in pigs and to measure the sulfide release from the sulfide releasing drug GYY4137 over 24h in cell culture medium. In addition this method reproducibly was used to determine the absorption of a spike of 100μM (34)S(2-) at pH 7.4 in buffer, blood, plasma, cysteine, glutathione and albumine. Baseline blood sulfide concentrations in pigs were between 0.2 and 2.1μM. Sulfide concentrations of a 300μM solution of GYY4137 in cell culture medium (in cell culture flask, gas exchange enabled) were 1.02μM (10min after GYY addition), 1.32μM (2h), 1.37μM (4h), 1.60μM (8h), 1.58μM (12h) and 1.81μM (24h). The sulfide concentration of the corresponding medium without GYY4137 ranged between 0.25 and 0.50μM. An initial 100μM spike of (34)S(2-) in blood decreased to 23μM after 1min, 12μM after 10 and 1μM after 60min. The corresponding values for plasma were 60, 41 and 3μM, for phosphate buffer (pH 7.4) 103, 96 and 85μM. In a 50mg/ml solution of albumine the (34)S(2-) concentration decreased to 22, 15, 3μM, in 5mg/ml oxidized glutathione to 32, 18, 3μM, in 5mg/ml reduced glutathione to 79, 76, 75μM, in 120μg/ml cystine to 91, 64, 12μM and in 1mg/ml cysteine to 110, 102, 99μM. Our findings show, that sulfide baseline concentrations in blood are in the low micromolar range. Added sulfide is rapidly absorbed by blood, plasma and disulfide bridge containing peptides and proteins, but much lower by the reduced species. Adding the sulfide releasing drug GYY4137, increased the sulfid concentration of cell culture media for 24h.
    No preview · Article · Sep 2013 · Nitric Oxide