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.

A comprehensive study of ovine haemostasis to assess suitability to model human coagulation.

INTRODUCTION: Similarities in size, anatomy and physiology have supported the use of sheep to model a wide range of human diseases, including coagulopathy. However, coagulation studies involving sheep are limited by the absence of high quality data defining normal ovine coagulation and fibrinolysis. MATERIALS AND METHODS: Full blood examination, routine and specialised coagulation tests, rotational thromboelastometry and whole blood platelet aggregometry was performed on 50 healthy Samm & Border Leicester Cross ewes and compared to corresponding human ranges. Intraspecies breed and gender variability was investigated by comparison to a smaller population of 13 healthy Merino wethers. RESULTS: Ovine coagulation was similar to human according to routine coagulation methods (PT, aPTT, TCT, Fib(C)) and some specialised coagulation tests (vWF, AT, Plasmin Inh). Despite these similarities, ovine secondary haemostasis demonstrated substantial differences to that of human. Rapid initiation of the contact activation pathway, high levels of FVIII, low Protein C, greater overall clot firmness and a reduced capacity for clot lysis was documented in sheep. In addition, ADP and collagen agonists precipitated a reduced primary haemostatic response in sheep relative to human. Intraspecies differences in whole blood platelet aggregometry between the cohorts of sheep indicate the need for breed-specific normal ranges. CONCLUSIONS: The application of a board spectrum of coagulation assays has enabled elucidation of the similarities as well as differences between ovine and human coagulation. The new knowledge generated from this study will guide the design of future translational coagulation studies in ovine models.