In our study, sheep were used as experimental animals. The heart structure, size and hemodynamics of sheep are similar to those of human. Using sheep as experimental animals, the experimental procedures are very close to clinical, and the experimental results have more guiding significance for clinical application. The character of sheep is docile, which is conducive to perioperative management. We established a long term “awake” extracorporeal membrane oxygenation model in both V-A and V-V ECMO mode in sheep. This system achieved long-term respiratory support or both respiratory and circulatory support. In our study, the ambulatory sheep obtained adequate nutrition from normal eating and maintained a satisfactory hemoglobin level, with no need of additional artificial nutritional support or blood transfusion.
V-V ECMO model was established cannulated by single site percutaneous DLC. The DLC withdraw total venous blood from both IVC and SVC through a drainage lumen for oxygenation, then, the oxygenated blood can be infused back to the RA. The drainage lumen openings in the SVC and IVC are spatially separated from the infusion lumen opening in the RA, which maximally reduces recirculation and enhances blood transfer efficiency [7,11,12]. V-V ECMO by DLC can provide long-term respiratory support with less invasive procedure and bleeding events while reducing the IMV-related complications.
Awake ECMO management is crucial for successful weaning and perioperative management is the key to the success of this model. Necropsy and pathological results also showed that the perioperative management strategies were effective. The anticoagulant management in the pilot study (3 sheep with V-V ECMO) referred to previous studies [7,8,13-15], ACT value was set at 180-220s. However, the fibrinogen and thrombosis formed in the oxygenator within 48 hours, which indicated that the sheep needed higher anticoagulant conditions. Meanwhile, bleeding signs were not observed, hemoglobin concentration and platelet count were relatively stable, so we adjusted the target ACT value to 220-250s in the formal experiment. Fibrinogen deposition and thrombosis formation in the oxygenator were reduced, oxygenation performances were stable, and no serious coagulation events occurred. However, it should be emphasized that the catheter should be placed cautiously during the operation, and the hemostasis and suture should be done carefully to avoid postoperative bleeding caused by the operation.
Respiratory monitoring was a major challenge in postoperative management. Physicians were blinded to both airway pressures and tidal volumes, which were main respiratory monitoring in ventilated patients. We should pay attention to avoid regurgitation and aspiration after spontaneous breathing recovery of the sheep. In our experiment, after the spontaneous breath recovered and the tracheal tube was removed, respiratory rate and blood gas analysis were monitored every 6 hours. The sweep gas flow and FiO2 of the oxygenator were adjusted according to the PaO2 and PaCO2. Therefore, we maintained PaCO2 at 35-40mmHg and PaO2 more than 80mmHg in the whole process of the experiment.
Fluid management during awake ECMO support was imperative. Some retrospective studies reported that fluid overload (FO) occurred commonly in patients supported with ECMO [16,17]. Progressive FO during the ECMO is associated with acute kidney injury (AKI), higher mortality, prolonged mechanical ventilation and ECMO duration. We paid close attention to the fluid balance (positive balance 800-1000ml per day) of the experimental sheep every day and maintained central venous pressure (CVP) at 5-12 cmH2O. No diuretic was used and the urine color was clear. The creatinine levels during ECMO support (115.04±19.83μmol/L) and after weaning (105.00±22.26μmol/L) were lower than the baseline level (134.44±15.20μmol/L), indicating that the renal tissue perfusion was good and the renal function was maintained in the normal range. In the temperature management of experimental sheep during ECMO support, according to the basic temperature of sheep (rectal temperature 39 ± 0.5℃), we set the temperature of the heat exchanger at 38.5 ℃. The rectal temperature of experimental sheep was maintained at 38-39.5℃.
Compared to the perioperative management of V-V ECMO, there are some key points in the management of V-A ECMO. Because V-A ECMO involves both arterial and venous systems, partly or completely replacing cardiopulmonary function, we paid more attention to anticoagulation and infection indexes. During the perioperative anticoagulation with heparin, the change of ACT was closely monitored, the dosage of heparin was adjusted in time, and the coagulation signs were closely observed. In terms of anti-infection management, in addition to the routine incision disinfection and application of antibiotics, we also closely monitored the changes of body temperature and infection indicators (such as white blood cell count and hsCRP).
The advantage of ambulation of the experimental sheep was obvious [4,6]. First of all, because the experimental sheep can breathe and cough spontaneously, the possibility of pulmonary infection was reduced. Second, no intubation allowed independent feed to get enough nutrition, contributing to normal hemoglobin level and no need for blood transfusion. In this study, consciousness and autonomous activities of sheep increased the difficulty of ECMO management. In order to ensure the stable flow and avoid cannula dislocation or kinking in awake animal, we restricted the sheep properly (Fig. 1C shows the details). No cannula kinking or displacement events appeared.
Patients receiving ECLS support may benefit from early mobilization. For instance, early mobilization may be particularly beneficial in patients awaiting heart or lung transplantation, as maintenance of physical conditioning may be an important component of a patient’s transplant candidacy. The ability to engage critically ill patients in active physical therapy and early mobilization necessarily involves minimization of sedation and is often further facilitated by a strategy that favors endotracheal extubation . Informed by reports of successful use, transplant centers are rapidly embracing awake ECMO as a means to maintain patient viability and survival to bridge-to-transplant (BTT). One center reported a six-month survival rate of 80% in patients supported on awake ECMO pre-transplant, compared to 50% of patients maintained on mechanical ventilation, which indicated ECMO support in patients who are awake and nonintubated represents a promising bridging strategy . Another experienced center reported survival rates of 84% at two years post-transplant for patients supported with ECMO prior to surgery, which demonstrated favorable survival in patients receiving awake ECMO as a bridge to lung transplantation . As a BTT strategy, awake ECMO can afford critically ill patients freedom from mechanical ventilation and can enable daily rehabilitation, including ambulation.
Previous studies have shown that the initiation of ECMO is associated with an immediate and complex inflammatory reaction, similar to that seen in systemic inflammatory response syndrome (SIRS). At that moment when the patient’s blood first comes into contact with the foreign surface of the extracorporeal circuit, a variety of coagulative and inflammatory cascades are activated. Levels of pro-inflammatory cytokines rise rapidly [20-22], which, in association with activation of the complement and contact systems, results in leukocyte activation . This innate immune response, if severe, persistent or unchecked by a compensatory anti-inflammatory response (CARS), may lead to endothelial injury, disrupted microcirculation, and end organ dysfunction [24,25]. Under the condition of good perfusion of main organs and tissues, according to the histopathological evaluation of our experiment, we found that the inflammatory reaction caused by ECMO circuit itself may be at a low level, it had little effect on the function of main organs and tissues in sheep. At the same time, it also showed that our perioperative anti-infection management was relatively successful.
To our knowledge, this study is the first awake large animal model to explore the long-term survival in both V-V and V-A ECMO simultaneously. According to our pre-experiment and previous clinical experience, we made a detailed management plan in advance and adjusted it dynamically during experimental progress. We optimized the perioperative management and made pathological analysis after the endpoint, focusing on the effect of long-term awake ECMO on tissues and organs. As the experimental sheep are healthy, according to the histopathological analysis after the endpoint, we were able to explore the effect of ECMO circuit itself on organs and tissues.
The establishment and management experience of long-term awake ECMO in big animal model will lay a solid foundation and provide a stable platform for further research. The establishment of long-term ECMO disease model (such as cardiogenic shock, heart failure and acute respiratory distress syndrome model) in large animals will be closer to the actual situation of clinical patients, but at the same time, the requirements of perioperative management are also higher. In previous studies, the disease models of ECMO in large animals were acute disease models, and the survival time of large animals was less than 24 hours [13-15,26-34]. Our study can provide technical support and management strategies for the establishment of long-term awake ECMO disease model in large animals in the future. For instance, in the future clinical practice, when the optimization or change of management strategy and concept is involved, but it is uncertain whether patients will get a profit, this model can be used for preclinical verification, and specific data such as pathophysiological changes of tissues and organs can be obtained. In addition, this study can also provide a stable platform for the development and optimization of extracorporeal life support equipment in the future. In general, our study has extensive clinical translational value.
Our study has several limitations. First, this research is still in the preliminary stage of long-term awake ECMO model in big animals and the number of experimental sheep was relatively small. Second, in order to keep the blood volume and hemoglobin concentration in the normal range, except for basic hematology and coagulation data, we did not dynamically monitor the levels and changes of inflammation related indicators. Third, there were two peaks in white blood cell count during the experiment, it is necessary to take blood culture and adjust the use of antibiotics. Further research is needed to standardize the establishment and management of long-term awake ECMO model, and focus on long-term ECMO support related application expansion, management strategy optimization, pathophysiology exploration, equipment development and subsequent establishment of the disease animal model.