Warenits, Alexandra-Maria; Sterz, Fritz; Schober, Andreas; Ettl, Florian; Magnet, Ingrid Anna Maria; Högler, Sandra; Teubenbacher, Ursula; Grassmann, Daniel; Wagner, Michael; Janata, Andreas; Weihs, Wolfgang

INTRODUCTION

Out-of-hospital sudden cardiac arrest (OOHCA) is a leading cause of death in developed countries ^(1–3). Conventionally treated CA is still associated with an unsatisfactory outcome ⁽⁴⁾. The use of extracorporeal life support (ECLS), in a selected population of patients with refractory CA, has become a potentially life-saving treatment option ^(5–10). ECLS is also used as rescue therapy in the setting of refractory cardiogenic shock, acute respiratory failure, or septic shock unresponsive to conventional therapeutic options. However, survival rates of these critically ill patients treated with ECLS are still improvable and there remains an urgent need for further research to advance this novel therapeutic strategy ^(11–13).

Animal models play an important role in translational ECLS research. It has been shown that porcine models are appropriate to investigate the use of ECLS in refractory shock and cardiac arrest ^(14–16). However, there exists no perfect animal model of the human cardiovascular system. Each model has advantages and disadvantages to be compared to the human situation. Rodent animal models are commonly used in cardiac research to perform proof-of-principle experiments rapidly and cost-efficiently, especially before performing experiments in large animals ⁽¹⁷⁾. A rat model of ECLS was established to investigate pathophysiological mechanism and further therapeutic modifications during ECLS to possibly improve the clinical use of cardiopulmonary bypass (CPB) ⁽¹⁸⁾. The major limiting factors in performing such resuscitation experiments in rat models are the complexity of the experimental procedures, and the requirement of well-trained experimenter microsurgical skills for the vessel preparation. Especially during the pilot phase of ECLS, rat models high complication and drop-out rates, due to serious adverse events (SAEs) are common. Only some experimental research groups report their observed SAEs during resuscitation experiments in rats ^(19–26). In our laboratory, we implemented an ECLS experiment in the rat based on an existing model ⁽²⁷⁾. During the pilot and the study phase of our ECLS experiments, we lost many animals due to SAEs. We report our observations and describe some possible solutions based on our institutional experience, to prevent others from avoidable SAEs in future CPB experiments and to reduce animal resources.

MATERIALS AND METHODS

The experimental protocol was performed with the approval of the Institutional Animal Care and Use Committee of the Medical University of Vienna and the Austrian Federal Ministry of Science, Research and Economy (GZ.: 66.009/0064-II/3b/2011) followed the ARRIVE ⁽²⁸⁾ and Directive 2010/63/EU guidelines. Two teams, consisting of two emergency medicine physicians with different experience in animal preparation, performed the experiments within 12 months with the support of a highly experienced veterinary physician. In this report, we analyzed 126 ECLS rat experiments, based on a common protocol. The experiments included pilot and study animals to describe all observed adverse events during model development. There were some differences concerning the chosen temperature management and survival time to the study endpoint in the different ECLS experiments, dependent on the individual pilot or study protocol. Our finally established ventricular fibrillation cardiac arrest (VFCA) model with ECLS resuscitation allowed us to collect hemodynamic, metabolic, and histopathological data. These findings compared to naive controls and sham-operated groups would go beyond the scope of this article and will be published elsewhere.

Animal preparation and cardiopulmonary bypass setup

Adult male Sprague-Dawley rats (350–600 g body weight [BW], 3-month old) were brought to the laboratory 14 days before the experiment (Fig. 1). The experimental protocol of all performed ECLS experiments consisted of six phases (Fig. 2). Briefly, all rats were sedated with sevoflurane, received analgesics, and were orotracheally intubated and mechanically ventilated. Continuous monitoring was established (see Table, Supplemental Digital Content 1, http://links.lww.com/SHK/A411, which describes the anesthesia and monitoring in detail). Using aseptic techniques, the left femoral vein and artery were cannulated via surgical cut-downs with a heparinized catheter (Argyle Polyurethane Umbilical Vessel Catheter; 2.5 Fr, Convidien, Mansfield, Mass). The catheters (see Image, Supplemental Digital Content 2, http://links.lww.com/SHK/A412, which illustrates the left femoral venous and arterial catheters) were advanced 9 cm and 13 cm into the left femoral artery and vein. They were used for venous drug and fluid administration, arterial blood sampling and for a continuous measurement of central venous pressure (CVP) and mean arterial pressure (MAP). The right femoral artery was cannulated with an arterial inflow bypass cannula (Dipl.Ing. Martin Humbs, Mitterdarching, Valley, Germany). The custom venous bypass cannula (14G × 50 mm, five-holes, along its distal two-thirds) (see Image, Supplemental Digital Content 3, http://links.lww.com/SHK/A413, which shows the venous bypass cannula) was placed in the cranially ligated right jugular vein, with the tip ending in the inferior vena cava. After removing the rigid metal insertion-stylet from the venous cannula, a human neonatal pacing catheter (Vygon GmbH & Co Bi-Pacing-ball 3 Fr, Aachen, Germany) was inserted to induce VFCA in the rat. A custom-made CPB circuit (Dipl.Ing. Martin Humbs, Mitterdarching, Valley, Germany) (see image, Supplemental Digital Content 4, http://links.lww.com/SHK/A414, which illustrates the components of the CPB circuit and image, Supplemental Digital Content 5, http://links.lww.com/SHK/A415, which demonstrates the experimental CPB setup) consisted of an open venous reservoir, a roller pump (Masterflex L S PTFE Tubing Pump, Cole-Parmer Instrument Co, Vernon Hills, Ill), silicone tubing, a membrane oxygenator (Dipl.Ing. Martin Humbs, Mitterdarching, Valley, Germany) and a heat exchanger with circulating water bath (VWR Polyscience Refrigerated/Heated 1166D Circulator Water Bath, Ill). The membrane oxygenator contained a disposable three-layer capillary membrane with a gas exchange area sufficient to provide a PaO₂ > 400 mm Hg. Heparin (1 I.U./mL crystalloid solution) was administered into the crystalloid priming solution (ELO-MEL isoton Infusionslösung, Fresenius Kabi GmbH, Graz, Austria).

Fig. 1

Fig. 2

Experimental protocol

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.

Fig. 3

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.

Study endpoint

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.

RESULTS

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.

Fig. 4

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.

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.

DISCUSSION

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.

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).

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.

Acknowledgments

The authors acknowledge all healthcare professionals, technicians of the Core Unit of Biomedical Research, Medical University of Vienna, and our students.

REFERENCES

Supplemental Digital Content

• SHK_00_00_2016_06_27_STERZ_SHOCK-D-16-00204_SDC1.docx; [Word] (84 KB)
• SHK_00_00_2016_06_27_STERZ_SHOCK-D-16-00204_SDC2.jpg; [Image] (414 KB)
• SHK_00_00_2016_06_27_STERZ_SHOCK-D-16-00204_SDC3.jpg; [Image] (6.48 MB)
• SHK_00_00_2016_06_27_STERZ_SHOCK-D-16-00204_SDC4.jpg; [Image] (322 KB)
• SHK_00_00_2016_06_27_STERZ_SHOCK-D-16-00204_SDC5.jpg; [Image] (2.2 MB)
• SHK_00_00_2016_06_27_STERZ_SHOCK-D-16-00204_SDC6.jpg; [Image] (327 KB)
• SHK_00_00_2016_06_27_STERZ_SHOCK-D-16-00204_SDC7.jpg; [Image] (6.34 MB)