Pre-clinical study protocol: Blood transfusion in endotoxaemic shock.

The Surviving Sepsis Campaign (SCC) and the American College of Critical Care Medicine (ACCM) guidelines recommend blood transfusion in sepsis when the haemoglobin concentration drops below 7.0 g/dL and 10.0 g/dL respectively, while the World Health Organisation (WHO) guideline recommends transfusion in septic shock 'if intravenous (IV) fluids do not maintain adequate circulation', as a supportive measure of last resort. Volume expansion using crystalloid and colloid fluid boluses for haemodynamic resuscitation in severe illness/sepsis, has been associated with adverse outcomes in recent literature. However, the volume expansion effect(s) following blood transfusion for haemodynamic circulatory support, in severe illness remain unclear with most previous studies having focused on evaluating effects of either different RBC storage durations (short versus long duration) or haemoglobin thresholds (low versus high threshold) pre-transfusion. •We describe the protocol for a pre-clinical randomised controlled trial designed to examine haemodynamic effect(s) of early volume expansion using packed RBCs (PRBCs) transfusion (before any crystalloids or colloids) in a validated ovine-model of hyperdynamic endotoxaemic shock.•Additional exploration of mechanisms underlying any physiological, haemodynamic, haematological, immunologic and tissue specific-effects of blood transfusion will be undertaken including comparison of effects of short (≤5 days) versus long (≥30 days) storage duration of PRBCs prior to transfusion.

Fluid resuscitation with 0.9% saline alters haemostasis in an ovine model of endotoxemic shock.

INTRODUCTION: Fluid resuscitation is a cornerstone of severe sepsis management, however there are many uncertainties surrounding the type and volume of fluid that is administered. The entire spectrum of coagulopathies can be seen in sepsis, from asymptomatic aberrations to fulminant disseminated intravascular coagulation (DIC). The aim of this study was to determine if fluid resuscitation with saline contributes to the haemostatic derangements in an ovine model of endotoxemic shock. MATERIALS AND METHODS: Twenty-one adult female sheep were randomly divided into no endotoxemia (n = 5) or endotoxemia groups (n = 16) with an escalating dose of lipopolysaccharide (LPS) up to 4 µg/kg/h administered to achieve a mean arterial pressure below 60 mmHg. Endotoxemia sheep received either no bolus fluid resuscitation (n = 8) or a 0.9% saline bolus (40 mL/kg over 60 min) (n = 8). No endotoxemia, saline only animals (n = 5) underwent fluid resuscitation with a 0.9% bolus of saline as detailed above. Hemodynamic support with vasopressors was initiated if needed, to maintain a mean arterial pressure (MAP) of 60-65 mm Hg in all the groups. RESULTS: Rotational thromboelastometry (ROTEM®) and conventional coagulation biomarker tests demonstrated sepsis induced derangements to secondary haemostasis. This effect was exacerbated by saline fluid resuscitation, with low pH (p = 0.036), delayed clot initiation and formation together with deficiencies in naturally occurring anti-coagulants antithrombin (p = 0.027) and Protein C (p = 0.001). CONCLUSIONS: Endotoxemia impairs secondary haemostasis and induces changes in the intrinsic, extrinsic and anti-coagulant pathways. These changes to haemostasis are exacerbated following resuscitation with 0.9% saline, a commonly used crystalloid in clinical settings.

Inflammation and lung injury in an ovine model of fluid resuscitated endotoxemic shock.

BACKGROUND: Sepsis is a multi-system syndrome that remains the leading cause of mortality and critical illness worldwide, with hemodynamic support being one of the cornerstones of the acute management of sepsis. We used an ovine model of endotoxemic shock to determine if 0.9% saline resuscitation contributes to lung inflammation and injury in acute respiratory distress syndrome, which is a common complication of sepsis, and investigated the potential role of matrix metalloproteinases in this process. METHODS: Endotoxemic shock was induced in sheep by administration of an escalating dose of lipopolysaccharide, after which they subsequently received either no fluid bolus resuscitation or a 0.9% saline bolus. Lung tissue, bronchoalveolar fluid (BAL) and plasma were analysed by real-time PCR, ELISA, flow cytometry and immunohistochemical staining to assess inflammatory cells, cytokines, hyaluronan and matrix metalloproteinases. RESULTS: Endotoxemia was associated with decreased serum albumin and total protein levels, with activated neutrophils, while the glycocalyx glycosaminoglycan hyaluronan was significantly increased in BAL. Quantitative real-time PCR studies showed higher expression of IL-6 and IL-8 with saline resuscitation but no difference in matrix metalloproteinase expression. BAL and tissue homogenate levels of IL-6, IL-8 and IL-1ß were elevated. CONCLUSIONS: This data shows that the inflammatory response is enhanced when a host with endotoxemia is resuscitated with saline, with a comparatively higher release of inflammatory cytokines and endothelial/glycocalyx damage, but no change in matrix metalloproteinase levels.

Unintended Consequences: Fluid Resuscitation Worsens Shock in an Ovine Model of Endotoxemia.

RATIONALE: Fluid resuscitation is widely considered a life-saving intervention in septic shock; however, recent evidence has brought both its safety and efficacy in sepsis into question. OBJECTIVES: In this study, we sought to compare fluid resuscitation with vasopressors with the use of vasopressors alone in a hyperdynamic model of ovine endotoxemia. METHODS: Endotoxemic shock was induced in 16 sheep, after which they received fluid resuscitation with 40 ml/kg of 0.9% saline or commenced hemodynamic support with protocolized noradrenaline and vasopressin. Microdialysis catheters were inserted into the arterial circulation, heart, brain, kidney, and liver to monitor local metabolism. Blood samples were recovered to measure serum inflammatory cytokines, creatinine, troponin, atrial natriuretic peptide, brain natriuretic peptide, and hyaluronan. All animals were monitored and supported for 12 hours after fluid resuscitation. MEASUREMENTS AND MAIN RESULTS: After resuscitation, animals that received fluid resuscitation required significantly more noradrenaline to maintain the same mean arterial pressure in the subsequent 12 hours (68.9 mg vs. 39.6 mg; P = 0.04). Serum cytokines were similar between groups. Atrial natriuretic peptide increased significantly after fluid resuscitation compared with that observed in animals managed without fluid resuscitation (335 ng/ml [256-382] vs. 233 ng/ml [144-292]; P = 0.04). Cross-sectional time-series analysis showed that the rate of increase of the glycocalyx glycosaminoglycan hyaluronan was greater in the fluid-resuscitated group over the course of the study (P = 0.02). CONCLUSIONS: Fluid resuscitation resulted in a paradoxical increase in vasopressor requirement. Additionally, it did not result in improvements in any of the measured microcirculatory- or organ-specific markers measured. The increase in vasopressor requirement may have been due to endothelial/glycocalyx damage secondary to atrial natriuretic peptide-mediated glycocalyx shedding.

Incremental research approach to describing the pharmacokinetics of ciprofloxacin during extracorporeal membrane oxygenation.

BACKGROUND: Significant interactions between drugs, extracorporeal membrane oxygenation (ECMO) circuits and critical illness may affect the pharmacokinetic properties of antibiotics in critically ill patients receiving ECMO. OBJECTIVE: To describe the pharmacokinetic properties of ciprofloxacin during ECMO by integrating pre-clinical findings (ie, ex vivo and in vivo ovine models) to a critically ill patient. DESIGN, PARTICIPANTS AND INTERVENTION: An ex vivo model of an ECMO circuit was used to describe ciprofloxacin concentration changes over 24 hours. An in vivo ovine model of ECMO was used to describe the population pharmacokinetic properties of ciprofloxacin in three different groups of sheep, and to investigate sources of pharmacokinetic variability. In the final phase, data from a 39-year-old critically ill man was used to validate the findings from the ovine pharmacokinetic model. RESULTS: In the ex vivo model of ECMO circuits, the median concentrations of ciprofloxacin at baseline and at 24 hours after ciprofloxacin infusion were similar. The time course of ciprofloxacin in the in vivo ovine on ECMO model was adequately described by a two-compartment model. The final population primary parameter mean estimates were: clearance (CL), 0.21 L/kg/h (SD, 0.09 L/kg/h) and volume of distribution (Vd), 0.84 L/kg (SD, 0.12 L/kg). In the critically ill ECMO patient, the primary pharmacokinetic parameter estimates were: CL, 0.15 L/kg/h and Vd, 0.99 L/kg. CONCLUSIONS: We provide preliminary evidence that ciprofloxacin dosing in ECMO patients should remain in line with the recommended dosing strategies for critically ill patients not receiving ECMO.

Evidence of altered haemostasis in an ovine model of venovenous extracorporeal membrane oxygenation support.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a life-saving modality used in the management of cardiopulmonary failure that is refractory to conventional medical and surgical therapies. The major problems clinicians face are bleeding and clotting, which can occur simultaneously. To discern the impact of pulmonary injury and ECMO on the host's haemostatic response, we developed an ovine model of smoke-induced acute lung injury (S-ALI) and ECMO. The aims of this study were to determine if the ECMO circuit itself altered haemostasis and if this was augmented in a host with pulmonary injury. METHODS: Twenty-seven South African meat merino/Border Leicester Cross ewes underwent instrumentation. Animals received either sham injury (n = 12) or S-ALI (n = 15). Control animal groups consisted of healthy controls (ventilation only for 24 h) (n = 4), ECMO controls (ECMO only for 24 h) (n = 8) and S-ALI controls (S-ALI but no ECMO for 24 h) (n = 7). The test group comprised S-ALI sheep placed on ECMO (S-ALI + ECMO for 24 h) (n = 8). Serial blood samples were taken for rotational thromboelastometry, platelet aggregometry and routine coagulation laboratory tests. Animals were continuously monitored for haemodynamic, fluid and electrolyte balances and temperature. Pressure-controlled intermittent mandatory ventilation was used, and mean arterial pressure was augmented by protocolised use of pressors, inotropes and balanced fluid resuscitation to maintain mean arterial pressure >65 mmHg. RESULTS: Rotational thromboelastometry, platelet aggregometry and routine coagulation laboratory tests demonstrated that S-ALI and ECMO independently induced changes to platelet function, delayed clot formation and reduced clot firmness. This effect was augmented with the combination of S-ALI and ECMO, with evidence of increased collagen-induced platelet aggregation as well as changes in factor VIII (FVIII), factor XII and fibrinogen levels. CONCLUSIONS: The introduction of an ECMO circuit itself increases collagen-induced platelet aggregation, decreases FVIII and von Willebrand factor, and induces a transient decrease in fibrinogen levels and function in the first 24 h. These changes to haemostasis are amplified when a host with a pre-existing pulmonary injury is placed on ECMO. Because patients are often on ECMO for extended periods, longer-duration studies are required to characterise ECMO-induced haemostatic changes over the long term. The utility of point-of-care tests for guiding haemostatic management during ECMO also warrants further exploration.

Inflammation and lung injury in an ovine model of extracorporeal membrane oxygenation support.

Extracorporeal membrane oxygenation (ECMO) is a life-saving treatment for patients with severe refractory cardiorespiratory failure. Exposure to the ECMO circuit is thought to trigger/exacerbate inflammation. Determining whether inflammation is the result of the patients' underlying pathologies or the ECMO circuit is difficult. To discern how different insults contribute to the inflammatory response, we developed an ovine model of lung injury and ECMO to investigate the impact of smoke-induced lung injury and ECMO in isolation and cumulatively on pulmonary and circulating inflammatory cells, cytokines, and tissue remodeling. Sheep receiving either smoke-induced acute lung injury (S-ALI) or sham injury were placed on veno-venous (VV) ECMO lasting either 2 or 24 h, with controls receiving conventional ventilation only. Lung tissue, bronchoalveolar fluid, and plasma were analyzed by RT-PCR, immunohistochemical staining, and zymography to assess inflammatory cells, cytokines, and matrix metalloproteinases. Pulmonary compliance decreased in sheep with S-ALI placed on ECMO with increased numbers of infiltrating neutrophils, monocytes, and alveolar macrophages compared with controls. Infiltration of neutrophils was also observed with S-ALI alone. RT-PCR studies showed higher expression of matrix metalloproteinases 2 and 9 in S-ALI plus ECMO, whereas IL-6 was elevated at 2 h. Zymography revealed higher levels of matrix metalloproteinase 2. Circulating plasma levels of IL-6 were elevated 1-2 h after commencement of ECMO alone. These data show that the inflammatory response is enhanced when a host with preexisting pulmonary injury is placed on ECMO, with increased infiltration of neutrophils and macrophages, the release of inflammatory cytokines, and upregulation of matrix metalloproteinases.

The impact of acute lung injury, ECMO and transfusion on oxidative stress and plasma selenium levels in an ovine model.

The purpose of this study was to determine the effects of smoke induced acute lung injury (S-ALI), extracorporeal membrane oxygenation (ECMO) and transfusion on oxidative stress and plasma selenium levels. Forty ewes were divided into (i) healthy control (n=4), (ii) S-ALI control (n=7), (iii) ECMO control (n=7), (iv) S-ALI+ECMO (n=8) and (v) S-ALI+ECMO+packed red blood cell (PRBC) transfusion (n=14). Plasma thiobarbituric acid reactive substances (TBARS), selenium and glutathione peroxidase (GPx) activity were analysed at baseline, after smoke injury (or sham) and 0.25, 1, 2, 6, 7, 12 and 24h after initiation of ECMO. Peak TBARS levels were similar across all groups. Plasma selenium decreased by 54% in S-ALI sheep (1.36±0.20 to 0.63±0.27µmol/L, p<0.0001), and 72% in sheep with S-ALI+ECMO at 24h (1.36±0.20 to 0.38±0.19, p<0.0001). PRBC transfusion had no effect on TBARS, selenium levels or glutathione peroxidase activity in plasma. While ECMO independently increased TBARS in healthy sheep to levels which were similar to the S-ALI control, the addition of ECMO after S-ALI caused a negligible increase in TBARS. This suggests that the initial lung injury was the predominant feature in the TBARS response. In contrast, the addition of ECMO in S-ALI sheep exacerbated reductions in plasma selenium beyond that of S-ALI or ECMO alone. Clinical studies are needed to confirm the extent and duration of selenium loss associated with ECMO.