This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.
Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.
Solid organ transplantation in general and cardiac transplantation, in particular, are on the rise worldwide1,2. The standard method of organ procurement is donation after brain death (DBD). Given the strict inclusion criteria of DBD, less than 40% of the offered hearts are accepted3, thereby limiting the offer in face of increasing demand and extending the organ waiting list. To address this issue, the use of organs donated after circulatory death (DCD) is considered a potential solution4.
In DCD donors, however, an agonal phase following withdrawal of care and a period of unprotected warm ischemia before resuscitation are inevitable5. The potential organ injury after circulatory death can lead to organ dysfunction, explaining the reluctance to routinely adopt DCD heart transplantations. It is reported that only 4 centers use DCD hearts clinically, with stringent criteria that includes very short warm ischemia times and young donors without chronic pathologies6,7. For ethical and legal reasons, limited or no cardioprotective interventions can be applied in donors prior to circulatory death5,8,9. Thus, any mitigation to alleviate the ischemia-reperfusion (IR) injury is limited to cardioprotective therapies initiated during early reperfusion with cardioplegic solutions, and do not allow for proper functional assessment. Ex vivo heart perfusion (EVHP) and reconditioning of the DCD heart using dedicated platforms has been proposed as an alternative solution and studied by various scholars10,11,12,13. EVHP offers a unique opportunity to deliver post-conditioning agents to DCD hearts to improve functional recovery. However, for efficient clinical translation, many technical and practical issues remain to be addressed, and this is further compounded by a lack of consensus on a range of perfusion and functional criteria to determine transplantability6,8.
Herein we report the development of a reproducible pre-clinical small animal DCD protocol combined with an ex vivo heart perfusion system that can be used to investigate organ post-conditioning initiated at the time of procurement, during initial reperfusion, and/or throughout EVHP.
All animal care and experimental protocols conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee of the Centre Hospitalier de l’Université de Montréal Research Center.
1. Preliminary Preparations
2. Animal Preparation
3. Initiation of Cardiac Donation After Circulatory Death (DCD) Protocol
NOTE: A complete protocol timeline can be seen in Figure 2.
4. Ex Vivo Heart Perfusion System (EVHP) and Cardiac Functional Assessment
5. End of Experience
6. Data analyses
Following extubation, blood pressure rapidly drops in a predictable pattern (Figure 3). Expected time to death is less than 5 min.
Figure 4 shows an average pressure/time curve at the start of reconditioning following 0, 10 and 15 min of WIT. Contractile function will improve over time. The use of short periods of WIT will allow for contractility to return to normal, and morphological damage will not be detectable (Figure 5 and Figure 6).
Proof-of-concept use of a conditioning agent added with the cardioplegia and at the stabilization phase show that the damage generated by 15 min of WIT in this model are amenable to modulation by cardioprotective agents (Figure 4, Figure 5 and Figure 6).
Figure 1: Required equipment schemas. Minimal requirements for a (A) cardioplegia delivery system and a (B) Langendorff ex vivo heart perfusion system. Please click here to view a larger version of this figure.
Figure 2: Protocol timeline. Timeline from the moment of extubation until the end of the protocol. In control animals, cardioplegia is initiated without DCD or warm ischemic time. Please click here to view a larger version of this figure.
Figure 3: Intracarotid blood pressure/time plot. Typical evolution of intracarotid blood pressure following extubation. Warm ischemia time stars when the peak systolic blood pressure drops below 30 mmHg, or if asystole or ventricular fibrillation appears, whatever comes first. Please click here to view a larger version of this figure.
Figure 4: Ex vivo average beat-to-beat ventricular pressure time curve. Image derived from analyses of data taken after 10 min stabilization and perfusion (time 0 in figure 2) with or without the use of an experimental pharmacological cardioprotective conditioning agent. Ischemic time refers to warm ischemic times (WIT). Please click here to view a larger version of this figure.
Figure 5: Ex vivo recovery and functional analyses. (A) Continuous ventricular pressure-time curve after 10 min stabilization and perfusion with or without the use of an experimental pharmacological cardioprotective conditioning agent. Arrows show artifacts due to manual modification of EDP. (B) Maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV vs. time plot derived from (A) showing a time-dependant improvement in contractility without treatment (green line). Short WIT (red line) or treated (yellow) hearts show a pattern similar to the control group (blue line). Data points are the mean of at least 200 individual beats. Bars show the standard error of the mean of each data point. Please click here to view a larger version of this figure.
Figure 6: 2,3,5-Triphenyl-tetrazolium chloride coloration at the end of experiments. Infarct area observed following diverse warm ischemic times (WIT) and the use of a pharmacological cardioprotective conditioning agent. Brick red: viable tissue. Light yellow: non-viable tissue. Please click here to view a larger version of this figure.
Supplementary Figure 1: Setup photograph. (A) Photograph showing the setup for the cardioplegia delivery system. Numbered equipment corresponds to: cardioplegia container (1), bubble trap (2), pressure sensor and catheter (3), peristaltic pump (4), polygraph connected to the pressure sensor (5) and small animal ventilator (6). (B) Photograph showing the setup for the Langendorff ex vivo heart perfusion system. Numbered equipment corresponds to: Perfusate container (1), conditioning agent container (2) and heart chamber (3). Please click here to download this figure.
Supplementary Figure 2: Neck dissection. (A) Photography showing the exposed jugular vein (arrow) prior to heparin injection. (B) shows the dissected carotid artery (arrow) with the sutures placed for bleeding control. Please click here to download this figure.
Supplementary Figure 3: Opening of the atria to prevent recirculation. (A) Photography showing the opening of the left atrial appendage (1). On the background the aorta (2) is clamped above the diaphragm (3). (B) Shows the opening of the right atrial appendage (1). Please click here to download this figure.
Supplementary Figure 4: Heart procurement. (A) Photography showing the use of curved forceps to separate the aorta (arrow) and the pulmonary artery. (B) Photography showing cardiac dissection and procurement. The heart is hold by the aorta using forceps. Please click here to download this figure.
The protocol presented here introduces a simple, convenient and versatile model of cardiac DCD, offering the opportunity to assess cardiac functional recovery, tissue damage and the use of post-conditioning cardioprotective agents to improve recovery of donor hearts otherwise discarded for transplantation. Ex vivo heart perfusion systems (EVHP) systems have been optimized to provide a platform for evaluating cardiac function and offer a unique opportunity to deliver and test modified solutions supplemented with post-conditioning pharmacological agents to preserve and repair DCD hearts in small15 and large animals16,17 models of cardiac DCD. Nevertheless the protocols are often insufficiently detailed and not always clinically relevant, making clinical translation difficult.
In the realm of DCD models, ex vivo DCD models, like the one described by Sanz18, lack an agonal phase. By inducing cardiac arrest by stopping mechanical ventilation, the sympathetic nervous system is overactivated, leading to a “catecholamine storm”19. This increase in catecholamines modifies the characteristics of the donor organs, and has been linked to a reduced functional status of experimental DCD organs19. Additionally, the progressive decline in function prior to asystole leads to right ventricular distension and consequent injury. In our protocol, we have induced circulatory death using a clinically relevant asphyxiation model, which maintains these responses.
Two main in vivo cardiac DCD models are described in the literature: open chest15 and closed chest20 models. Cardiac physiology is altered by the open chest approach by reducing the mechanical lung/heart interaction and preload. Furthermore, in open chest procedures, body heat loss is accelerated, further affecting functional outcomes21. Therefore it is preferable to maintain a closed chest approach preventing heat loss. Another refinement is to minimize the variability of time to circulatory death. Kearns et al. reported that time to death (time to non-pulsatile or mean blood pressure less than 30 mmHg) was between 3 to 11 min. In the 10 and 20 min WIT, 40% and 60% of hearts did not recover function, respectively, on an ex vivo working heart apparatus, making data interpretation more difficult15. An alternative to reduce the time to circulatory death is to use paralytic agents20; nevertheless, some evidence points towards direct cardiac effects of vecuronium, due to its effects on sympathetic and parasympathetic innervation22. To increase reproducibility, we elected for tracheal clamping, combined with a precise arterial pressure monitorization, allowing for a more homogeneous agonal time (<5 min). It is known that organ damage starts before the moment of circulatory death; with some authors considering a cut-off systolic blood pressure below 50 mmHg as the beginning of functional WIT6, explaining the reluctance to transplant organs following a long period form withdraw of life sustaining measures until reperfusion. In this protocol, the WIT definition used follows the current experimental standard15, nevertheless, further studies are needed to clarify the exact set of hemodynamic parameters that mark the induction of organ damage in order to improve WIT calculation, thus offering better information for clinical practice.
The infusion of cardioplegic solution at constant physiological pressure and temperature offers a unique opportunity to initiate heart conditioning and tissue protection with any pharmacological agent or by other means. Technical refinements include clamping the thoracic aorta, limiting perfusion to the heart and thereby reducing the amount of solution needed for each essay. Once the heart is on the EVHP system, standardized functional evaluation is necessary. It has been shown that the use of an EVHP system has the potential to improve resuscitation of hearts previously considered not transplantable23,24. Interestingly, the clinically available EVHP system evaluates cardiac viability only by using serial lactate measurements8,23. Lactate measurements are not related to cardiac performance of DCD hearts24,25, thus additional measurements to evaluate transplantability are necessary. This experimental setup allows for a complete functional evaluation, including generated pressures and myocardial contractility measurements including +dP/dT and –dP/dT, allowing for a more thorough evaluation of cardiac function before the final transplant decision is made. Additionally, measurements of cardiac troponin, a marker of myocardial damage directly correlated to ischemic infarct size26, and release kinetics are related to the extent of cardiac ischemia in a Langendorff ischemia/reperfusion system. In particular, with long ischemic times (60 min), troponin levels are maintained after 1 h reperfusion, while LDH and creatinine kinase significantly decrease, and being non related to the extent of cardiac damage27,28, thus the use of serial troponin measures ensure a complete evaluation of organ viability before transplant. A major confounding variable in cardiac functional evaluation is the heart rate. Spontaneous heart rate is inversely related to length of ischemia29, and heart rate directly correlates with +dP/dt in isolated rat hearts30 and in animal models31. Interestingly, in recently published work on rodent models of DCD hearts and EVHP conditioning, pacing was not used and cardiac rates were variable and recorded in their protocols15,18,20. To maintain physiological heart rate, pacing was used once the heart had recovered rhythmic contraction. The chosen 300 bpm frequency is similar to those of healthy, non-stressed rats32.
Limitations of this protocol include the use of volatile anesthetic for induction. These agents have been shown to confer ischemic preconditioning33. Nevertheless, the short time of inhaled anesthetic use had no observable effect in this protocol and progressive myocardial dysfunction was still noted with increasing WIT. The use of normothermic cardioplegia can be also viewed as a limitation. Using normothermic cardioplegia allows optimal translation from the in vitro conditions used for the development of pharmacological conditioning agents, since cells are usually maintained at 37 °C. Nevertheless, in this setup cardioplegia temperature can be easily regulated according to the requirements of the investigator. On the other hand, the use of a Langendorff preparation versus a working heart preparation for reconditioning might also be seen as a limitation. The working heart preparation allows for continuous recording of a pressure/volume loop12,15, with controlled pre and afterload, allowing for complete functional evaluation. The main advantage of a Langendorff preparation is that it maintains a constant aortic and perfusion pressure, especially during initial reperfusion, when generated pressure is minimal. In addition, the evaluation setup is simpler for the Langendorff heart compared to a working heart preparation. Nevertheless, this setup can be converted into a working heart preparation if deem necessary. Alternatively, cardiac reanimation can be performed in situ using normothermic regional perfusion, with cardiac performance being measured directly by the use of a Millar catheter34, allowing comprehensive hemodynamic and myocardial functional evaluation before organ procurement. In humans, both in situ and ex vivo reconditioning strategies have been described6, thus the development of both models allows for experimental comparisons that may translate in to optimization of clinical practice. Finally, the small size and high heart rate of this animal model may be considered as a limitation due to the potential technical difficulties observed while performing these experiments, and the inevitable physiological differences between rat and human hearts. If the EVHP evaluation is already standardized, a researcher can be familiarized with this technique by performing as little as 3 experiments. On the other hand, the use of this small animal model allows for convenient screening at a reasonable cost, reserving larger and more costly animal models such as the porcine model, to therapies with high human translational potential.
In conclusion, the protocol described here takes into account the best practices emanating from several groups researching DCD hearts. This protocol grants full control of WIT, allowing for a comprehensive structural and functional evaluation of cardioprotective conditioning treatment strategies in rats. This protocol can be upscaled and transferred to large animal models, allowing to translate research findings to clinical reality and ultimately allowing development of novel therapies increasing the quality and availability of lifesaving organs much needed by patients.
The authors have nothing to disclose.
Portions of this work were supported by a generous contribution by the Fondation Marcel et Rolande Gosselin and Fondation Mr Stefane Foumy. Nicolas Noiseux is scholar of the FRQ-S.
The authors wish to thanks Josh Zhuo Le Huang, Gabrielle Gascon, Sophia Ghiassi, and Catherine Scalabrini for their support in data collection.
0,9% Sodium Chloride. 1L bag | Baxter | Electrolyte solution for flushing in the modified Langendorff system. | |
14G 2" I.V catheter | Jelco | 4098 | To act as endotracheal tube. |
2,3,5-Triphenyltetrazolium chloride | Milipore-Sigma | T8877 | Vital coloration |
22G 1" I.V catheter | BD | 383532 | I.V catheter with extension tube that facilitates manipulation for carotid catheterization |
Adson Dressing Fcp, 4 3/4", Serr | Skalar | 50-3147 | Additional forceps for tissue manipulation |
Alm Self-retaining retractor 4×4 Teeth Blunt 2-3/4" | Skalar | 22-9027 | Tissue retractor used to maintain the chest open. |
Bridge amp | ADinstruments | FE221 | Bridge amp for intracarotid blood pressure measurement |
Calcium chloride | Milipore-Sigma | C1016 | CaCl2 anhydrous, granular, ≤7.0 mm, ≥93.0% Part of the Krebs solution |
D-(+)-Glucose | Milipore-Sigma | G8270 | D-Glucose ≥99.5% Part of the Krebs solution |
DIN(8) to Disposable BP Transducer | ADinstruments | MLAC06 | Adapter cable for link between bridge amp and pressure transducer |
Disposable BP Transducer (stopcock) | ADinstruments | MLT0670 | Pressure transducer for intracarotid blood pressure measurement |
dPBS | Gibco | 14190-144 | Electrolyte solution without calcium or magnesium. |
Eye Dressing Fcp, Str, Serr, 4" | Skalar | 66-2740 | Additional forceps for tissue manipulation |
Formalin solution, neutral buffered, 10% | Milipore-Sigma | HT501128 | Fixative solution |
Heating Pad | Sunbean | 756-CN | |
Heparin sodium 1000 UI/mL | Sandoz | For systemic anticoagulation | |
Hydrochloric Acid 36,5 to 38,0% | Fisher scientific | A144-500 | Diluted 1:1 for pH correction |
Ketamine | Bimeda | Anesthetic. 100 mg/ml | |
LabChart | ADinstruments | Control software for the Powerlab polygraph, allowing off-line analyses. Version 7, with blood pressure and PV loop modules enabled | |
Left ventricle pressure balloon | Radnoti | 170404 | In latex. Size 4. |
Lidocaine HCl 2% solution | AstraZeneca | Antiarrhythmic for the cardioplegic solution | |
Magnesium Chloride ACS | ACP Chemicals | M-0460 | MgCl2+6H2O ≥99.0% Part of the Krebs solution |
Micro pressure sensor | Radnoti | 159905 | Micro pressure sensor and amplifier connected to the intraventricular balloon |
Pacemaker | Biotronik | Reliaty | Set to generate a pulse each 200 ms for a heart rate of 300 bpm. |
pH bench top meter | Fisher scientific | AE150 | |
Physiological monitor | Kent Scientific | Physiosuite | For continuous monitoring of rodent temperature and saturation during the procedure |
Plasma-Lyte A | Baxter | Electrolyte solution used as base to prepare cardioplegia | |
Potassium Chloride | Milipore-Sigma | P4504 | KCl ≥99.0% Part of the Krebs solution |
Potassium Chloride 2 meq/ml | Hospira | Part of the cardioplegic solution | |
PowerLab 8/30 Polygraph | ADinstruments | Electronic polygraph | |
Silk 2-0 | Ethicon | A305H | Suture material for Langendorff apparatus |
Silk 5-0 | Ethicon | A302H | Suture material for carotid |
Small animal anesthesia workstation | Hallowell EMC | 000A2770 | Small animal ventilator |
Sodium bicarbonate | Milipore-Sigma | S5761 | NaHCO3 ≥99,5% Part of the Krebs solution |
Sodium Chloride | Milipore-Sigma | S7653 | NaCl ≥99.5% Part of the Krebs solution |
Sodium Hydroxide pellets | ACP chemicals | S3700 | Diluted to 5N (10 g in 50 ml) for pH correction |
Sodium phosphate monobasic | Milipore-Sigma | S0751 | NaH2PO4 ≥99.0% Part of the Krebs solution |
Stevens Tenotomy Sciss, Str, Delicate, SH/SH, 4 1/2" | Skalar | 22-1240 | Small scisors for atria and cava vein opening |
Tissue slicer blades | Thomas scientific | 6727C18 | Straight carbon steel blades for tissue slicing at the end of the protocol |
Tuberculin safety syringe with needle 25G 5/8" | CardinalHealth | 8881511235 | For heparin injection |
Veterinary General Surgery Set | Skalar | 98-1275 | Surgery instruments including disection scisors and mosquito clamps |
Veterinary Micro Set | Skalar | 98-1311 | Surgery instruments with microscisors used for carotid artery opening |
Working Heart Rat/Guinea Pig/Rabbit system | Radnoti | 120101BEZ | Modular working heart system modified for the needs of the protocol. Includes all the necesary tubbing, water jacketed reservoirs and valves, including 2 and 3 way stop cock |
Xylazine | Bayer | Sedative. 20 mg/ml |