Porcine Sepsis Research Articles

Continuous exhaled breath carbon isotope analysis for early detection of sepsis

Approximately 750,000 people develop sepsis each year with more than 215,000 deaths. Earlier diagnosis and treatment of sepsis is critical to improving outcome.The exhaled breath delta value (i.e. 13CO2/12CO2 ratio) is a biomarker for the acute phase response (APR) to endotoxin. The objective of this study was to demonstrate that the breath delta value is a leading marker for the onset of infection in a porcine sepsis model.Sepsis was induced by cecal ligation and puncture. Briefly, pigs were anesthetized and positive pressure ventilation was established. The animal was instrumented for invasive blood pressure monitoring, cardiac output measurements, and blood sampling. Heart rate, respiratory rate, arterial blood pressure, central venous pressure, blood oxygen saturation, temperature, blood glucose, end tidal CO2, and breath delta value were monitored once every 15 minutes.Sepsis caused an increase in body temperature between 6 and 15 hours after onset. Other physiological parameters did not change from baseline until vascular hypotension was observed and cardiac parameters deteriorated. The breath delta value decreased from baseline 6 to 10 hours after onset and preceded changes in body temperature, cardiac and vascular parameters by a minimum of 4 hours.These data show that the breath delta value is a leading indicator of infection in peritoneal sepsis when compared to other physiological parameters.Research supported by a Wisconsin Innovation and Economic Development Research Grant.

Combined inhibition of complement (C5) and CD14 markedly attenuates blood thrombogenicity, inflammation and hemodynamic changes in porcine sepsis

Combined inhibition of complement (C5) and CD14 markedly attenuates blood thrombogenicity, inflammation and hemodynamic changes in porcine sepsis

The Role of Bradykinin and the Effect of the Bradykinin Receptor Antagonist Icatibant in Porcine Sepsis

Bradykinin (BK) is regarded as an important mediator of edema, shock, and inflammation during sepsis. In this study, we evaluated the contribution of BK in porcine sepsis by blocking BK and by measuring the stable BK metabolite, BK1-5, using anesthetized pigs. The effect of BK alone, the efficacy of icatibant to block this effect, and the recovery of BK measured as plasma BK1-5 were first investigated. Purified BK injected intravenously induced an abrupt fall in blood pressure, which was completely prevented by pretreatment with icatibant. BK1-5 was detected in plasma corresponding to the doses given. The effect of icatibant was then investigated in an established model of porcine gram-negative sepsis. Neisseria meningitidis was infused intravenously without any pretreatment (n = 8) or pretreated with icatibant (n = 8). Negative controls received saline only. Icatibant-treated pigs developed the same degree of severe sepsis as did the controls. Both groups had massive capillary leakage, leukopenia, and excessive cytokine release. The plasma level of BK1-5 was low or nondetectable in all pigs. The latter observation was confirmed in supplementary studies with pigs undergoing Escherichia coli or polymicrobial sepsis induced by cecal ligation and puncture. In conclusion, icatibant completely blocked the hemodynamic effects of BK but had no beneficial effects on N. meningitidis-induced edema, shock, and inflammation. This and the fact that plasma BK1-5 in all the septic pigs was virtually nondetectable question the role of BK as an important mediator of porcine sepsis. Thus, the data challenge the current view of the role of BK also in human sepsis.

Sepsis, Kidney, and Tissue Oxygenation

Sepsis, Kidney, and Tissue Oxygenation

Assessments of skin and tongue microcirculation reveals major changes in porcine sepsis

To examine the relation between central hemodynamics, clinical severity and microvascular findings in tongue and skin during sepsis. Skin and tongue microcirculation was examined using laser Doppler and video microscopy techniques before and 200 min after inducing sepsis in pigs (n=6) by inactivated Neisseria meningitides and in two control animals. All infected pigs developed clinical signs of sepsis. Pericapillary bleedings developed in the tongue in the two pigs with the most severe disease. Capillary density increased in the groin skin in infected pigs after 200 min as compared to baseline (P<0·02). In the same period, mean capillary flow velocity was reduced in groin skin and tongue in septic pigs (P<0·02). At 200 min a fraction of capillaries had developed 'no flow' or 'brisk flow', patterns hardly seen at baseline. Laser Doppler perfusion was reduced in ear and tongue after 200 min (P<0·02 for both). The described pathology was more pronounced in the pigs with the most severe sepsis. Capillary bleedings may be used as an early indication of severe sepsis. Examination of skin and tongue microcirculations may be used to characterize severity of sepsis and possibly to assess effect of treatment.

LLAMA HEAVY-CHAIN ANTIBODY FRAGMENTS EFFICIENTLY REMOVE TOXIC SHOCK SYNDROME TOXIN 1 FROM PLASMA IN VITRO BUT NOT IN EXPERIMENTAL PORCINE SEPTIC SHOCK

Staphylococcus aureus produces the superantigen toxic shock syndrome toxin 1 (TSST-1). When the bacterium invades the human circulation, this toxin can induce life-threatening gram-positive sepsis. Current sepsis treatment does not remove bacterial toxins. Variable domains of llama heavy-chain antibodies (VHH) against toxic shock syndrome toxin 1 ([alpha]-TSST-1 VHH) were previously found to be effective in vitro. We hypothesized that removing TSST-1 with [alpha]-TSST-1 VHH hemofiltration filters would ameliorate experimental sepsis in pigs. After assessing in vitro whether timely removing TSST-1 interrupted TSST-1-induced mononuclear cell TNF-[alpha] production, VHH-coated filters were applied in a porcine sepsis model. Clinical course, survival, plasma interferon [gamma], and TSST-1 levels were similar with and without VHH-coated filters as were TSST-1 concentrations before and after the VHH filter. Plasma TSST-1 levels were much lower than anticipated from the distribution of the amount of infused TSST-1, suggesting compartmentalization to space or adhesion to surface not accessible to hemofiltration or pheresis techniques. Removing TSST-1 from plasma was feasible in vitro. However, the [alpha]-TSST-1 VHH adsorption filter-based technique was ineffective in vivo, indicating that improvement of VHH-based hemofiltration is required. Sequestration likely prevented the adequate removal of TSST-1. The latter warrants further investigation of TSST-1 distribution and clearance in vivo.

Effects of inhaled carbon monoxide and glucocorticoids in porcine endotoxin sepsis

Recent animal studies have demonstrated that pre-treatment with inhaled carbon monoxide (iCO) exert anti-inflammatory effects in various septic models. In all these models, there is no information whether iCO might act therapeutically after the onset of septic damage. The objective of this study was to investigate the potential anti-inflammatory effects of iCO to treat established injury in a model of porcine endotoxin sepsis. Five groups of pigs (n=6 in each group), were studied under anesthesia and mechanical ventilation: healthy control group (HC); lipopolysaccharide (LPS) groups, animals received continuous IV infusion of LPS for 6 hours; 2.5 hours after of LPS infusion treated groups received either: 250 ppm of iCO for 3.5 h, (LPS+CO group); 3 mg/Kg hydrocorti-sone bolus [Steroid (ST)], (LPS+ST group); or both steroid and iCO, (LPS+CO+ST group). Measurements of haemodynamics, blood gases, respiratory mechanics and biochemistry of organ function, were made. At the end of the experiment lung tissue was taken for analysis of histology and inflammatory markers: tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), activator protein-1 (AP-1) and glucocorticoid receptor (GR). LPS administration induced a dramatic inflammatory injury in lungs, increased expression of TNF-α, NF-κB, AP-1, down regulation of GR, pulmonary hypertension and severe deterioration of respiratory mechanics, oxygenation and organ function. Treatment with steroids and to greater extent with iCO significantly improved the microscopic appearance of the lung but had no effect on inflammatory markers. iCO significantly decreased pulmonary hypertension induced by LPS, without an obvious protective effect on organ function. Using this porcine sepsis model we find that treatment with iCO after the septic damage decreases pulmonary hypertension and partially protects the lung tissue from the inflammatory destruction induced by LPS but has no beneficial effects on organ function.

EFFECTS OF COMBINING INDUCIBLE NITRIC OXIDE SYNTHASE INHIBITOR AND RADICAL SCAVENGER DURING PORCINE BACTEREMIA

Complex interactions of nitric oxide and other free radicals have been implicated in the pathogenesis of sepsis and organ dysfunction. We hypothesized that simultaneous inducible nitric oxide synthase inhibition (L-N6-[1-iminoethyl]-lysine [L-NIL]) and neutralization of superoxide (O2-) (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl [Tempol]) would protect from detrimental consequences of long-term, volume-resuscitated, hyperdynamic porcine bacteremia. In this prospective, randomized, controlled experimental study, 16 anesthetized, mechanically ventilated and instrumented pigs were exposed to 24 h of continuous infusion of live Pseudomonas aeruginosa. After 12 h of hyperdynamic sepsis, animals were randomized to receive either vehicle (control, n = 8) or combination of L-NIL and Tempol (n = 8). Systemic and hepatosplanchnic hemodynamics, oxygen exchange, metabolism, ileal mucosal microcirculation and tonometry, oxidative stress and coagulation parameters were assessed before, 12, 18, and 24 h of P. aeruginosa infusion. Combined treatment inhibited sepsis-induced increase in plasma nitrate/nitrite, 8-isoprostane, and thiobarbituric acid reactive species concentrations, prevented hypotension, and reversed hyperdynamic circulation. Despite lower intestinal macrocirculation, combined regimen attenuated the otherwise progressive deterioration in ileal mucosal microcirculation and prevented mucosal acidosis. Treatment substantially attenuated mesenteric and hepatic venous acidosis, preserved sepsis-induced impairment of hepatosplanchnic redox state, and prevented the development of renal dysfunction. Finally, coinfusion of L-NIL and Tempol largely attenuated the sepsis-induced rise in plasma von Willebrand factor and thrombin-antithrombin complexes. Thus, hemodynamic, microcirculatory, metabolic, renal, and coagulation data indicate that combining inducible inhibition with cell permeable O2(-) radical scavenger afforded significant protection in porcine sepsis, thus suggesting an important interactive role of O2(-) and nitric oxide in mediating organ dysfunction.

CHEMICALLY MODIFIED TETRACYCLINE (COL-3) IMPROVES SURVIVAL IF GIVEN 12 BUT NOT 24 HOURS AFTER CECAL LIGATION AND PUNCTURE

Sepsis can result in excessive and maladaptive inflammation that is responsible for more than 215,00 deaths per year in the United State alone. Current strategies for reducing the morbidity and mortality associated with sepsis rely on treatment of the syndrome rather than prophylaxis. We have been investigating a modified tetracycline, COL-3, which can be given prophylactically to patients at high risk for developing sepsis. Our group has shown that COL-3 is very effect at preventing the sequelae of sepsis if given before or immediately after injury in both rat and porcine sepsis models. In this study, we wanted to determine the "treatment window" for COL-3 after injury at which it remains protective. Sepsis was induced by cecal ligation and puncture (CLP). Rats were anesthetized and placed into five groups: CLP (n = 20) = CLP without COL-3, sham (n = 5) = surgery without CLP or COL-3, COL3@6h (n = 10) = COL-3 given by gavage 6 h after CLP, COL3@12h (n = 10) = COL-3 given by gavage 12 h after CLP, and COL3@24h (n = 20) = COL-3 given by gavage 24 h after CLP. COL-3 that was given at 6 and 12 h after CLP significantly improved survival as compared with the CLP and the CLP@24h groups. Improved survival was associated with a significant improvement in lung pathology assessed morphologically. These data suggest that COL-3 can be given up to 12 h after trauma and remain effective.

INTERACTION OF POLYMICROBIAL SEPSIS AND O2 BREATHING ON HBO-INDUCED O2?? RADICAL PRODUCTION IN ISOLATED LYMPHOCYTES

Introduction: Exposure to hyperbaric oxygen (HBO) increases the formation of reactive oxygen species (ROS). Sepsis per se also enhances ROS formation, but it is unknown whether sepsis and/or O2 breathing also affect HBO-induced ROS formation. Therefore we analyzed the effect of ventilation with 100 % O2 on HBO-related ROS formation during porcine sepsis. Methods: Before as well as 12 and 24 hours after induction of fecal peritonitis, lymphocytes were isolated and exposed to HBO (2 hours at 4 bar). In cells incubated with the spin probe CMH, superoxide radical (O2−) concentrations were measured before and immediately after HBO exposure using electron spin resonance. Results: O2− production prior to HBO exposure progressively increased from 0.9 (0.1-1.5) before to 1.6 (0.3-13.4) nmol/min/106cells at 24 hours of peritonitis, resulting in a higher HBO-related rise (to 1.3 (0.5-4.2) before and 4.5 (1.2-13.3) nmol/min/106 cells at 24 hours of sepsis; p <.05). O2 breathing had no further effect (before HBO: from 1.0 (0.2-2.5) before sepsis to 1.8 (0.4-7.6) nmol/min/106 cells at 24 hours; after HBO: from 1.8 (0.8-8.8) before to 4.6 (0.9-9.8) nmol/min/106 cells at 24 hours). Conclusions: 24 hours of ventilation with 100 % O2 is safe with respect to the sepsis-related rise in HBO-induced lymphocyte O2− release. Supported by the Alexander-von-Humboldt-Stiftung and the Landesgraduiertenförderung Baden-Württemberg.

Endothelial and coagulation dysfunction during porcine bacteremia: effects of combining iNOS inhibitor and radical scavenger

We showed recently that both selective iNOS inhibition and the radical scavenger Tempol prevented live bacteria from causing key features of hemodynamic, microcirculatory and metabolic derangements in porcine sepsis [1,2]. Here we investigated the effects of combined selective iNOS inhibition (L-NIL) with a free radical scavenger (Tempol) on P. aeruginosa-induced endothelial and hemostatic stress.

Up-regulation of the glucocorticoid receptor and enhancement of the anti-inflammatory effects of steroids by inhaled nitric oxide in a porcine sepsis model

Up-regulation of the glucocorticoid receptor and enhancement of the anti-inflammatory effects of steroids by inhaled nitric oxide in a porcine sepsis model

Early signs of critical illness polyneuropathy in porcine sepsis

Early signs of critical illness polyneuropathy in porcine sepsis

Protein-free phospholipid emulsion treatment improved cardiopulmonary function and survival in porcine sepsis.

Lipoprotein phospholipid (PL) plays a major role in neutralization of endotoxin. This study tested the hypothesis that prophylactic administration of a PL-enriched emulsion (PRE), which augments PL content of serum lipoproteins and neutralizes endotoxin in vitro, would preserve cardiovascular function and improve survival in porcine septic peritonitis. A control group was compared with low-, mid-, and high-dose treatment groups that received PRE by primed continuous infusion for 48 h. A fibrin clot containing live Escherichia coli 0111.B4 was implanted intraperitoneally 30 min after the priming dose. Survival increased in a dose-dependent manner and was correlated with serum PL. Infused PL was associated with high-density lipoprotein in the low-dose group and all serum lipoproteins at higher doses. Treatment significantly lowered serum endotoxin and tumor necrosis factor (TNF)-alpha, preserved cardiac output and ejection fraction, and attenuated increases in systemic and pulmonary vascular resistances. This study demonstrated that augmentation of lipoprotein PL via administration of PRE improved survival and offered a novel therapeutic approach to sepsis.

Chronic Porcine Two-Hit Model with Hemorrhagic Shock and Pseudomonas aeruginosa Sepsis

Background: Sepsis is still a major cause of death despite well-developed therapeutical strategies such as antibiotics and supportive medication. The aim of this study was to characterize the long-term effects of a two-hit porcine sepsis model with a hemorrhagic shock as ‘first hit’ followed by a Pseudomonas aeruginosa infusion as ‘second hit’. Materials and Methods: Twelve juvenile healthy pigs were anesthetized and hemodynamically monitored. The two-hit group (n = 6) underwent a hemorrhagic shock with a 50% reduction of the mean arterial pressure and/or cardiac index for 45 min, followed by resuscitation, while the control group (n = 6) received no pretreatment. All chronically catheterized conscious pigs were challenged with a P. aeruginosa infusion (1.6 × 10⁷ CFU/kg/h for the first 24 h followed by 1.6 × 10⁶ CFU/kg/h for the next 24 h) and observed for another 48 h. Results: The two-hit group showed the following significant differences to the control group: higher APACHE II scores prior to sepsis induction, increased persisting mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) during bacterial challenge. In contrast, systemic vascular resistance (SVRI) was reduced at the end of the study. Throughout the observation period, the mean arterial pressure (MAP) was significantly reduced. Conclusions: The present study shows that the clinical course and hemodynamic effects of a P. aeruginosa sepsis will be aggravated by a preceding hemorrhagic shock during an oberservation period of 96 h. This two-hit model represents a valid, clinically relevant experimental protocol in sepsis research.

Treatment of porcine sepsis with high-dose antithrombin III reduces tissue edema and effusion but does not increase risk for bleeding.

We evaluated the effectiveness of antithrombin III (AT III) infusions designed to achieve supraphysiologic plasma levels of this serine protease inhibitor in preventing vascular permeability and disseminated intravascular coagulation in a pig model of sepsis. In addition, we determined whether high AT III doses were associated with increased bleeding risk. Sepsis was induced in 18 pigs by injection of lipopolysaccharide (LPS) (0.25 microg/kg per h for 3 h). At 90 min after the start of LPS infusion, pigs were randomized (n = 6 per group) to receive either human serum albumin as a placebo, AT III 120/5 (120 U/kg, 30-min bolus + 5 U/kg per h for 240 min), or AT III 250/10 (250 U/kg + 10 U/kg per h). Three additional animals served as negative controls (no LPS, no AT III). Treatment with AT III significantly reduced the amount of effluents in body cavities and fibrin monomers. AT III did not significantly increase bleeding risk as determined by organ hemorrhage. An additional assessment of AT III's bleeding risk [skin bleeding time (SBT)] was carried out in 35 nonseptic pigs treated with either AT III alone (120/5 or 250/10) or in the combination with heparin. Heparin administration alone produced a dose-dependent increase in SBT, but AT III alone did not. Addition of AT III 120/5 to heparin did not induce a further increase in bleeding time over heparin alone. These results indicate that administration of AT III in doses designed to achieve very high plasma concentrations significantly ameliorates symptoms of sepsis-induced vascular leakage and disseminated intravascular coagulation without increasing bleeding risk.

Interventions to improve cardiopulmonary hemodynamics during laparoscopy in a porcine sepsis model

Background: Laparoscopy is increasingly used in severely ill and acutely septic patients. In animals undergoing laparoscopy, the hemodynamic response to sepsis is blunted. Specific interventions to augment the hemodynamic potential may make laparoscopic intervention a safer alternative in septic patients. We compared different interventions to improve hemodynamic performance during exploratory laparoscopy in a porcine endotoxic shock model. Study Design: Domestic pigs (n = 12) received intravenous lipopolysaccharide injection and underwent surgical abdominal exploration using either laparoscopy or conventional laparotomy. For comparison, pigs exposed to endotoxin underwent laparoscopy with these interventions: intravenous infusions of prostacyclin (n = 5) or indomethacin (n = 4), intravenous crystalloid resuscitation (n = 5), pulmonary hyperventilation (n = 4), or abdominal insufflation with air (n = 5). Hemodynamic measurements and blood gas analyses were obtained using Swan-Ganz and arterial catheters. Results: Septic animals treated with prostacyclin undergoing laparoscopy had a higher cardiac index (CI, p < 0.01), stroke volume (SV; p < 0.001) and oxygen delivery (p < 0.05) than the untreated group. Likewise, treatment with indomethacin was associated with a higher CI (p < 0.001), SV (p < 0.005), and oxygen delivery (p < 0.005) compared with the untreated group. These effects may be secondary to a decreased pulmonary vascular resistance, demonstrated in the animals that received either prostacyclin (p < 0.05) or indomethacin (p < 0.05). In addition, animals given aggressive fluid resuscitation had a significantly higher CI (p < 0.05) and SV (p < 0.001) than those with normal fluid resuscitation during laparoscopy. Manipulation of arterial pH by insufflation of the abdomen with air to create the pneumoperitoneum, or by aggressively hyperventilating the animals, did not improve CI. Conclusions: Adverse effects of laparoscopy on cardiovascular hemodynamics in the septic state may be mediated by increased pulmonary vascular resistance, diminished venous return, or both. Specific interventions to reverse these variables may ameliorate hemodynamic changes seen.

Selective Downregulation of Neutrophils by a Phosphatidic Acid Generation Inhibitor in a Porcine Sepsis Model

Effects of lisofylline (1-(5-R-hydroxyhexyl)-3,7-dimethylxanthine), a functional inhibitor of phosphatidic acid (PA) generation derived fromde novosynthesis, on neutrophil function were examined in a porcine sepsis model. Hanford minipigs (18–25 kg) were randomly separated into six groups of six animals each: (1) saline control group; (2) sepsis control group, infused withPseudomonas aeruginosa(1 × 106colony-forming units/kg/min) for 2 h; (3) lisofylline control group, given a 25 mg/kg bolus of lisofylline 30 min prior to time zero, followed by a continuous infusion of 10 mg/kg/h throughout the study; (4) lisofylline pretreatment sepsis group, given lisofylline 30 min prior to sepsis, (5) lisofylline 1-h post-treatment sepsis group, and (6) lisofylline 2-h post-treatment sepsis group. All animals were studied for 6 h. Neutrophils were isolated at −0.5, 2, and 6 h. In the pretreatment and 1-h post-treatment groups, sepsis-induced neutrophil attachment to fibronectin was significantly attenuated. Sepsis-enhanced phagocytic activity was significantly reduced in the lisofylline pretreatment sepsis group, but not in the post-treatment groups. No treatment affected phorbol 12-myristate 13-acetate-induced chemiluminescence and basal filamentous actin content, which increased in sepsis, and cap formation, which declined in sepsis. Sepsis caused neutropenia, pretreatment produced neutrophilia, and 1-h post-treatment caused the neutropenia to recover to control levels. Interestingly, toward the end of the 6-h period, the neutrophil count was higher in the lisofylline control group than in the saline control groups. Thus, the inhibition of PA generation fromde novosynthesis during sepsis not only can selectively downregulate some neutrophil functions but can also reverse neutropenia.

Influence of dopexamine on leukocyte adherence and vascular permeability during endotoxemia in rat mesenteric venules

Dopexamine is a β2-adrenoceptor agonist with dopamine 1 and 2 receptor properties. It was recently shown that dopexamine was able to preserve the hepatic ultrastructure in a porcine sepsis model [1]. It seemed likely that the anti-inflammatory properties of dopexamine were associated with β2-adrenoceptor properties. The adherence of leukocytes to the vascular endothelium is an important step in the development of sepsis. The objective of this study was to investigate whether pretreatment with dopexamine could attenuate leukocyte adherence to venular endothelium and influence vascular permeability in post-capillary venules of the rat mesentery during endotoxemia.

Fatal myocardial depression and circulatory collapse associated with complement activation induced by plasma infusion in severe porcine sepsis

We have previously reported that fresh frozen plasma (FFP) may induce a rapid irreversible shock when repeatedly infused in pigs challenged with Gram-negative sepsis. The aims of the present study were to elucidate the cardiovascular nature of the shock and determine the aetiologic role of tumour necrosis factor (TNF), complement activation and halothane anaesthesia. Three groups of anaesthetized piglets were inoculated with a lethal dose of live E. coli bacteria. Groups I (n = 8) and III (n = 8) were anaesthetized with halothane and group II (n = 8) with ketamine. Animals in groups I and II received repeated infusions of FFP, whereas animals in group III received repeated infusions of 7% albumin. Six animals in group I and four animals in group II died during the first plasma infusion. Survival time was significantly longer in group II (P = 0.04) compared to group I. No animals in group III died during the albumin infusions, and no adverse effects were observed during the infusions. In group I the plasma induced shock was characterized by abruptly falling mean arterial pressure, cardiac index, systemic vascular resistance index and left ventricular contractility. Concomitant increases were recorded in left ventricular filling pressure and central venous pressure. Group II demonstrated a similar, but delayed response. Plasma infusion was associated with a significant increase in terminal complement complex (TCC) (P < 0.03 in group I, P < 0.05 in group II) and depletion of serum ionized calcium. We conclude that FFP may induce fatal myocardial depression and circulatory collapse in severe sepsis. Complement activation may be of aetiologic importance.