Preparation phase 1
The first baseline blood gas (analyzed for PaCO2, PaO2, pH, BE, sodium, potassium, chloride, bicarbonate, hematocrit, and lactate) was taken from the left femoral artery, and the monitored baseline hemodynamic parameters were noted (heart rate, MAP, CVP, etCO2, SpO2, and temperature) and the blood gas values were adapted to a physiological state if necessary. Before the insertion of the arterial and venous bypass cannulas, heparin (unfractionated heparin 500 I.U./kg BW) was administered intravenously to prevent clotting of the cannulas and catheters. After completion of all surgical procedures the second arterial blood gas baseline analysis was drawn to guarantee physiological conditions before the experiments began. Heating sources were removed and sedation with sevoflurane 3.5% was discontinued 90 s before current impulse was given via the pacing catheter placed in the right ventricle. Size-adapted self-adhesive paddles (Pediatric Plus Pads, Plug Style, for Heart-Start MRx ALS Monitor/Defibrillator, Philips Medical Systems HSG, Andover, Mass) were stitched to the shaven right and left lateral thorax and connected to a biphasic manual defibrillator (Heart-Start MRx ALS Monitor/Defibrillator, Philips Medical Systems HSG).
Cardiac arrest phase 2
Ten seconds before the initiation of VFCA mechanical ventilation was stopped (Fig. 3). VFCA was induced with an alternating current (AC) impulse of 12 V/50 Hz, at a maximum of 8 mA for 120 s via the pacing catheter. A maximum of 150 s of AC was delivered if spontaneous defibrillation occurred. At 180 s of CA the fibrillation catheter was removed and the venous cannula was connected to the bypass circuit. During the 10 min of untreated CA epinephrine (20 μg/kg BW), heparin (100 I.U.) and sodium bicarbonate (1 mmol/kg BW) were added to the venous reservoir.
Cardiopulmonary bypass phase 3
When 10 min of VFCA were reached, ECLS was initiated (Fig. 3). Mechanical ventilation (respiratory rate 20–40/min, FiO2 1.0) and CPB with an initial flow rate of 30 mL/min were initiated. The bypass flow rate was quickly increased to 100 mL/kg BW/min, with 100% oxygen at a gasflow of 100 mL/min to the oxygenator. At 75 s of resuscitation, epinephrine (10 μg/kg BW) was added to the reservoir and was then given intravenously at a 2-min interval. Temperature management during the resuscitation phase and after ROSC depended on the CPB study protocol. The circulating water bath temperature was set to 42°C priming and maintenance to reach normothermia, 30°C priming and 35°C maintenance for mild hypothermia and 11°C priming and 22°C maintenance for deep hypothermia during the CPB phase. Fluid boluses of crystalloid solution of 0.1 mL (maximum 10 mL) were administered intravenously to reach an MAP of 60 mm Hg. At 2 min of ECLS rats were defibrillated using a sequence of up to two 5-Joules of biphasic shocks, repeated every 2 min until ROSC was achieved. The attempted resuscitation was terminated, if ROSC was not achieved within 10 min of resuscitation.
Return of spontaneous circulation phase 4
When a stable hemodynamic status (MAP > 60 mm Hg) was present, CPB flow rate was decreased gradually for bypass weaning and mechanical ventilation was changed to initial state. Arterial blood gas samples were taken 5 and 15 min after ROSC.
Explantation phase 5
To preserve hemodynamic stability the venous jugular bypass cannula was removed first. All catheters were explanted, the vessels ligated, and the wounds sutured. Rats successfully weaned from mechanical ventilation were extubated and after an observational period transferred to the stable.
Intensive care phase 6
Rats were separately housed until full recovery with free access to food and water for the first 1 to 3 days after sustained ROSC. Analgesia was maintained as long as pain was present. If rats were unconscious in the first hours of recovery period, they received oxygen via a nose cone mask. Rodents presenting with weakness were fed by hand and received subcutaneous fluid if necessary. The core temperature was controlled (temperature was depending on randomized study group) with an intraperitoneal implanted E-Mitter telemetric transponder (TA E-Mitter, PDT-4000, Mini Mitter, A Respironics Inc Company, Bend, Ore), dedicated by a monitor with feedback mechanism to a warming or cooling ventilator for temperature management up to 12 h after ROSC. Weight and neurologic outcome were assessed daily for 2 weeks using the Overall Performance Category (OPC) score (29) and the Neurological Deficit Score (NDS) (30). If rats were presenting with a strongly reduced general condition, such as OPC 3 and 4, severe organ dysfunction, or weight loss of more than 20% despite adequate treatment and hand feeding, they were sacrificed.
Depending on the individual CPB study, animals survived between 5 and 12 weeks until study end. The rats were than euthanized, using an overdose of sevoflurane and further subcutaneously applied analgesics, and perfused transcardially with natrium chlorid 0.9% solution to remove all blood cells from the brain.
Availability of data and materials
Alexandra-Maria Warenits, the first author and Fritz Sterz, the corresponding author had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The data supporting the findings can be delivered on request.
Of the total 126 performed ECLS experiments, 68 (54%) rats achieved ROSC; 19 of these 68 rats died spontaneously and 4 moribund rats were sacrificed prior to the end of the study. Taking into account all animals included in the study, 22 survived until study endpoint, with a good neurological status (Fig. 4). Excellent functional neurologic long-term outcome (OPC 1) was found in 12 rats and minor neurological deficits (OPC 2) were found in 10.
The ECLS experiments were performed by two different teams, consisting of two emergency physicians with the support of a highly experienced veterinary physician within 12 months. The first team had experience in performing a conventional CPR rat model. The second team started the ECLS model with nearly no experience in small animal preparation.
Serious adverse events
A serious adverse event (SAE) was defined as an untoward occurrence during the experiment, which resulted in a life-threatening condition or death of the animal. A total of 76 SAEs happened during all performed 126 ECLS experiments. In 62 rats the observed SAEs were related to the experimental procedures (“REP”). These events originated in most cases due to problems of used techniques or unforeseeable equipment failures occurring during the experimental phases. In 14 cases of all observed SAEs, avoidable human errors (“goofs”) caused a life-threatening condition or death of the animal. Figure 4 gives an overview of the SAE incidence in each study phase and the time point and reason why the animals dropped out before reaching the study endpoint. The incidence and detailed description of all SAEs observed in each experimental phase are shown in Table 1.
Preparation phase 1
During the first experimental phase bleeding (n = 37) and cannula dislocation (n = 1) were observed as SAEs (n = 38). Due to human error, there was an accidental removal of the implanted femoral catheters which caused a lethal bleeding (human error, n = 4). The 34 cases of REPs were caused by hematothorax, resulting from tissue or vessel injuries during the insertion of the venous bypass cannula or the fibrillation catheter (REP, n = 27). The insertion of the arterial and venous femoral catheters caused lethal blood loss (REP, n = 6). In one case the venous bypass cannula was dislocated, ending with the tip in the pulmonary artery (REP, n = 1).
Cardiac arrest phase 2
In the CA phase bleeding (REP, n = 2) and material defect (REP, n = 1) occurred, related to experimental procedures. Despite a placed tourniquet on the venous jugular bypass cannula, containing the fibrillation catheter, lethal venous bleeding could not be avoided during the induction of CA in two animals. In one case a defect fibrillation catheter was exchanged and the replacement of the new catheter caused a lethal hematothorax.
Cardiopulmonary bypass phase 3
The observed SAEs (n = 12) during the CPB phase were bleeding, no achieved venous bypass drainage, too low bypass flow, material defects, and no oxygenation during CPB. Due to human error, accidental removal of the implanted arterial bypass cannula happened and caused a lethal bleeding in two rats (human error, n = 2). Bypass tubes (see Image, Supplemental Digital Content 6, http://links.lww.com/SHK/A416, visualizing the used CPB tubing) too small in diameter were accidentally used and resulted in a too low bypass flow in one case (human error, n = 1). In two experiments the oxygen tube was not connected to the bypass oxygenator (human error, n = 2). Venous bypass drainage could not be achieved in two animals, caused by a clot in the venous bypass cannula (REP, n = 2). In five experiments unpredictable material defects such as a rupture of a bypass tube, blood leak from oxygenator, and air bubbles present in the bypass system resulted in early termination of the experiments (REP, n = 5).
ROSC phase 4
After achieving ROSC, two SAEs occurred in all experiments due to human errors (human error, n = 2). In one rat air was sucked in the venous bypass cannula during accidental disconnection from the bypass tube causing lethal air embolism. Mechanical ventilation was discontinued in one animal, due to unnoticed disconnection of the endotracheal tube.
Explantation phase 5
Bleeding and air embolism were the observed SAEs during the explantation phase (SAE, n = 14). Lethal venous bleeding, from the left femoral vein (REP, n = 1) and the right jugular vein (REP, n = 3), was observed during the catheter and cannula explantation. In one case the venous catheter was accidentally removed from the femoral vein (human error, n = 1). During the explantation of the venous bypass cannula from the right jugular vein, air was sucked in through the holes of the tube and caused death in eight rats due to air embolism (REP, n = 8). Due to material defects, a venous bypass cannula was disconnected and caused lethal air embolism (REP, n = 1).
Intensive care phase 6
During the intensive care phase temperature management failures and serious diseases occurred as SAEs (n = 7). Due to human errors, the temperature management system was set to a wrong target temperature, and caused undesired lethal hyperthermia or hypothermia in the rats (human error, n = 2). Three animals died after development of severe peritonitis, without responding to empiric antimicrobial treatment (REP, n = 3). In two rats necrotic hind paws were observed (REP, n = 2).
Four animals with reduced general conditions (OPC 3 and OPC 4) were sacrificed during the intensive care phase. Two of the animals lost more than 20% of their body weight and had a strongly reduced general condition after the experiment. Histology confirmed purulent epidydimitis and gastric ulcers in one of these animals. Further two animal experiments were terminated because of weak general conditions, which were due to severe myocardial damage found during histological examination. Mild-to-moderate myocardial damage was present in all animals examined by histology.
We aimed to report and analyze all observed SAEs, during the implementation of our extracorporeal life support (ECLS) model in rats. In 76 of 126 experiments SAEs occurred: 62 (82%) were related to the experimental procedures (“REP”) and 14 (18%) were due to avoidable human errors (“goofs”). This number seems high under rigorous experimental conditions, and would by no means have been acceptable in the clinical context. Body mass dissimilarities and ECLS initiation prior to cardiac arrest would make it unreasonable to translate our results into clinical practice. However, after mastering all technical challenges these rodent models of VFCA with ECLS resuscitation will be useful to investigate new cardiopulmonary bypass treatment strategies. After analyzing all SAEs, we tried to find solutions for avoiding them and formulated some recommendations.
Serious adverse events related to experimental procedure (REP)
The highest incidence for REP occurring in the CPB experiments was found in the (1) preparation phase (n = 34) and in the (5) explantation phase (n = 13). The design and handling of the venous bypass cannula turned out to be a major obstacle to experimental success. During the preparation phase 49% of all observed SAEs happened. This was related to the high number of REP (n = 25) caused by lethal hematothorax during the insertion of the jugular venous bypass cannula we used in these CPB studies. This cannula contained a metal stylet with a sharp tip, which injured the thin tissue of the heart or the vena cava inferior during the insertion in all observed cases. In the explantation phase, eight of the observed 13 REP were directly related to technical problems concerning the venous bypass cannula. The cannula had openings for blood drainage all the way through the entire cannula side, which sucked air in the vessel during the explantation. We tried to minimize this risk of air embolism using different techniques for cannula removal. In the beginning, we connected a water filled syringe to the open end of the bypass cannula during explantation, without success in any cases. Later, we tilted the operation table about 45° and quickly removed the cannula and ligated the jugular vein simultaneously. All these described techniques could not completely avoid the risk of air embolism, through the drainage openings, of the venous bypass cannula. For future ECLS experiments we finally were able to develop a modified bypass cannula (see Image, Supplemental Digital Content 7, http://links.lww.com/SHK/A417, which shows the modified venous bypass cannula and the modified insertion-stylet). This cannula had five 5-mm-long side-openings, at a distance of 2.5 mm in the first third (2.5 cm) of the tube only. The proximal part was free from openings, which gave more room for safe insertion and explantation of the cannula, without causing a decrease of blood drainage during running bypass. The modified insertion-stylet had an atraumatic tip to minimize the risk of lethal injuries during insertion. After removing the stylet of the safely placed venous cannula and simultaneously clamping the proximal third, the fibrillation catheter with an integrated closing-cone (Combi-Stopper closing-cones, BBraun Melsungen AG, Melsungen, Germany) was inserted via the distal end after removing the clamp, and screwed to the proximal end of the bypass cannula. With this new method blood loss and air embolism could be minimized during insertion and explantation of the venous bypass cannula and the fibrillation catheter. Equipment failure of the bypass conduit, such as tube rupture, has been minimized by using it only once without autoclaving and reusing it. This method was more expensive but prevented unnecessary animal waste. We were able to formulate some recommendation concerning the solution of technical adverse events, as highlighted in Table 2.
Skill-based errors were mostly related to the preparation of the small vessels and caused lethal bleedings, especially in the early period of establishment. This necessary learning curve is a known phenomenon in small animal models containing surgical procedures (31–33). Inexperienced team members have to be intensely instructed by a highly experienced person. It is important to avoid changing team members during a CPB study to retain the learning curve and reduce complications in such a sensible experimental model. Retaining learned technical skills is negatively influenced by breaks in the experimental phase, like weekends or holidays. After such breaks the rate of observed complications and unsuccessful resuscitation is higher in the first attempts than when practiced frequently.
During 1 year of establishing an ECLS experiment, the team changed after the third quarter. In both teams 68% of the experiments were SAEs demanding study exclusion during the first 3 months. In the first team the occurrence of SAEs could be reduced in the second quarter to 40% of performed experiments. After three long experimentation breaks (20 days, 16 days, and 11 days) due to holidays, the occurrence of SAEs in the third quarter increased again to 55% (Fig. 5).
From our institutional experience, longer breaks and changes of the team have to be avoided. The best way to conduct such experiments is in one long run, to get a routine. We therefore recommend planning such complex experiments without a team change during the study phase. In addition, new team members should be trained by the old ones for at least 2 months, before starting as full team member. They should train their preparation techniques in at least 20 pilot animals, beginning with rats weighing 400 to 600 g is recommended, to exercise surgery skills in larger vessels.
Serious adverse events caused by human errors (“goof”)
Among all SAEs, 14 human errors were preventable. Errors of omission during the experimental procedures were observed as cause for such preventable SAEs. The implementation of detailed checklists for all procedural phases could have avoided these failures. In the future, we are going to adapt our existing checklists for all human errors that occurred over time to minimize such preventable SAEs.
Following the ARRIVE Guidelines from the National Centre for the Replacement, Refinement and Reduction of Animals in the Research, it is highly recommended to report in all detail occurred adverse events and the modifications in the experimental protocols, to minimize risk of error (28). If all resuscitation research groups would consequently report all complications and adverse events occurring during pilot and study phase of CPB experiments, the aim to refine and reduce the “wasting” of animals could be achieved.
To our knowledge, detailed reports about SAEs and their incidence in establishing an ECLS model in rats are missing in the state-of-the-art literature. SAEs are common in implementation of complex resuscitation models in rodents and should generally be reported in detail to avoid errors in future experiments. Knowing the source of possible errors from the literature helps to develop fault management prior to the commencement of the experimental study. Additional detailed online resources should be made available. This might help to work more efficiently due to reduction of rodents and costs in experimental CPB animal studies.
The authors acknowledge all healthcare professionals, technicians of the Core Unit of Biomedical Research, Medical University of Vienna, and our students.
1. Becker LB, Smith DW, Rhodes KV. Incidence of cardiac arrest
: a neglected factor in evaluating survival rates. Ann Emerg Med
1993; 22 1:86–91.
2. Atwood C, Eisenberg MS, Herlitz J, Rea TD. Incidence of EMS-treated out-of-hospital cardiac arrest
in Europe. Resuscitation
2005; 67 1:75–80.
3. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR, de Ferranti S, Després JP, Fullerton HJ, et al. Heart disease and stroke statistics-2015 update: a report from the American Heart Association. Circulation
2014; 131 4:e29–322.
4. Berdowski J, Berg RA, Tijssen JGP, Koster RW. Global incidences of out-of-hospital cardiac arrest
and survival rates: systematic review of 67 prospective studies. Resuscitation
2010; 81 11:1479–1487.
5. Cardarelli MG, Young AJ, Griffith B. Use of extracorporeal membrane oxygenation for adults in cardiac arrest
(E-CPR): a meta-analysis of observational studies. ASAIO J
2009; 55 6:581–586.
6. Fagnoul D, Taccone FS, Belhaj A, Rondelet B, Argacha J-F, Vincent JL, Backer DD. Extracorporeal life support associated with hypothermia and normoxemia in refractory cardiac arrest
2013; 84 11:1519–1524.
7. Ishaq M, Pessotto R. Might rapid implementation of cardiopulmonary bypass in patients who are failing to recover after a cardiac arrest
potentially save lives? Interact Cardiovasc Thorac Surg
2013; 17 4:725–730.
8. Gaieski DF, Boller M, Becker LB. Emergency cardiopulmonary bypass: a promising rescue strategy for refractory cardiac arrest
. Crit Care Clin
2012; 28 2:211–229.
9. Shin TG, Choi J-H, Jo IJ, Sim MS, Song HG, Jeong YK, Song YB, Hahn JY, Choi SH, Gwon HC, et al. Extracorporeal cardiopulmonary resuscitation
in patients with inhospital cardiac arrest
: a comparison with conventional cardiopulmonary resuscitation
. Crit Care Med
2011; 39 1:1–7.
10. Chen YS, Lin JW, Yu HY, Ko WJ, Jerng JS, Chang WT, Chen WJ, Huang SC, Chi NH, Wang CH, et al. Cardiopulmonary resuscitation
with assisted extracorporeal life-support versus conventional cardiopulmonary resuscitation
in adults with in-hospital cardiac arrest
: an observational study and propensity analysis. Lancet
2008; 372 9638:554–561.
11. de Waha S, Fuernau G, Eitel I, Desch S, Thiele H. Long-term prognosis after extracorporeal life support in refractory cardiogenic shock: results from a real-world cohort. EuroIntervention
2016; 11 12:1363–1371.
12. Beurtheret S, Mordant P, Paoletti X, Marijon E, Celermayer DS, Léger P, Pavie A, Combes A, Leprince P. Emergency circulatory support in refractory cardiogenic shock patients in remote institutions: a pilot study (the cardiac-RESCUE program). Eur Heart J
2013; 34 2:112–120.
13. Maclaren G, Butt W. Extracorporeal membrane oxygenation and sepsis. Crit Care Resusc
2007; 9 1:76–80.
14. Cherry BH, Nguyen AQ, Hollrah RA, Olivencia-Yurvati AH, Mallet RT. Modeling cardiac arrest
and resuscitation in the domestic pig. World J Crit care Med
2015; 4 1:1–12.
15. Weihs W, Krizanac D, Sterz F, Hlavin G, Janata A, Sipos W, Holzer M, Losert UM, Behringer W. Rapid induction of hypothermia with a small volume aortic flush during cardiac arrest
in pigs. Am J Emerg Med
2012; 30 5:643–650.
16. Sipos W, Duvigneau C, Sterz F, Weihs W, Krizanac D, Bayegan K, Graf A, Hartl R, Janata A, Holzer M, Behringer W. Changes in interleukin-10 mRNA expression are predictive for 9-day survival of pigs in an emergency preservation and resuscitation model. Resuscitation
2010; 81 5:603–608.
17. Milani-Nejad N, Janssen PML. Small and large animal models in cardiac contraction research: advantages and disadvantages. Pharmacol Ther
2014; 141 3:235–249.
18. Ali AA, Downey P, Singh G, Qi W, George I, Takayama H, Kirtane A, Krishnan P, Zalewski A, Freed D, et al. Rat model of veno-arterial extracorporeal membrane oxygenation. J Transl Med
19. Han F, Boller M, Guo W, Merchant RM, Lampe JW, Smith TM, Becker LB. A rodent
model of emergency cardiopulmonary bypass resuscitation with different temperatures after asphyxial cardiac arrest
2010; 81 1:93–99.
20. Bendel S, Springe D, Pereira A, Grandgirard D, Leib SL, Putzu A, Schlickeiser J, Jakob SM, Takala J, Haenggi M. Do different anesthesia regimes affect hippocampal apoptosis and neurologic deficits in a rodent cardiac arrest
model? BMC Anesthesiol
2015; 15 1:2.
21. Chen M-H, Liu T-W, Xie L, Song F-Q, He T, Zeng Z, Mo S-R. A simpler cardiac arrest
model in rats
. Am J Emerg Med
2007; 25 6:623–630.
22. Knapp J, Bergmann G, Bruckner T, Russ N, Böttiger BW, Popp E. Pre- and postconditioning effect of Sevoflurane on myocardial dysfunction after cardiopulmonary resuscitation
2013; 84 10:1450–1455.
23. Knapp J, Heinzmann A, Schneider A, Padosch SA, Böttiger BW, Teschendorf P, Popp E. Hypothermia and neuroprotection by sulfide after cardiac arrest
and cardiopulmonary resuscitation
2011; 82 8:1076–1080.
24. Ye S, Weng Y, Sun S, Chen W, Wu X, Li Z, Weil MH, Tang W. Comparison of the durations of mild therapeutic hypothermia on outcome after cardiopulmonary resuscitation
in the rat. Circulation
2012; 125 1:123–129.
25. Drabek T, Fisk JA, Dixon CE, Garman RH, Stezoski J, Wisnewski SR, Wu X, Tisherman SA, Kochanek PM. Prolonged deep hypothermic circulatory arrest in rats
can be achieved without cognitive deficits. Life Sci
2007; 81 7:543–552.
26. Samarska IV, Henning RH, Buikema H, Bouma HR, Houwertjes MC, Mungroop H, Struys MM, Absalom AR, Epema AH. Troubleshooting the rat model of cardiopulmonary bypass: effects of avoiding blood transfusion on long-term survival, inflammation and organ damage. J Pharmacol Toxicol Methods
2013; 67 2:82–90.
27. Janata A, Drabek T, Magnet IA, Stezoski JP, Janesko-Feldman K, Popp E, Garman RH, Tisherman SA, Kochanek PM. Extracorporeal versus conventional cardiopulmonary resuscitation
after ventricular fibrillation cardiac arrest
: a feasibility trial. Crit Care Med
2013; 41 9:e211–222.
28. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol
2010; 8 6:e1000412.
29. Xiao F, Safar P, Radovsky A. Mild protective and resuscitative hypothermia for asphyxial cardiac arrest
. Am J Emerg Med
1998; 16 1:17–25.
30. Katz L, Ebmeyer U, Safar P, Radovsky A, Neumar R. Outcome model of asphyxial cardiac arrest
. J Cereb Blood Flow Metab
1995; 15 6:1032–1039.
31. Hutchens MP, Traystman RJ, Fujiyoshi T, Nakayama S, Herson PS. Normothermic cardiac arrest
and cardiopulmonary resuscitation
: a mouse model of ischemia-reperfusion injury. J Vis Exp
32. Jungwirth B, Kellermann K, Qing M, Mackensen GB, Blobner M, Kochs EF. Cerebral tumor necrosis factor alpha expression and long-term neurocognitive performance after cardiopulmonary bypass in rats
. J Thorac Cardiovasc Surg
2009; 138 4:1002–1007.
33. Habertheuer A, Kocher A, Laufer G, Petzelbauer P, Andreas M, Aharinejad S, Ehrlich M, Wiedemann D. Innovative, simplified orthotopic lung transplantation in rats
. J Surg Res
2013; 185 1:419–425.
Cardiac arrest; cardiopulmonary bypass model; cardiopulmonary resuscitation; rats; rodent; serious adverse events
Supplemental Digital Content
© 2016 by the Shock Society