Pre-clinical Model of Cardiac Donation after Circulatory Death
August 2nd, 2019
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.
This protocol targets the limited transplantation organ supply issue by facilitating the evaluation of different cardioprotective conditioning strategies to allow the use of hearts donated after circulatory death. Place the animal on a heating pad set to medium. After confirming a lack of response to toe pinch in an anesthetized adult rat, insert a rectal temperature probe.
Attach a transdermal pulse oximeter sensor to a hind paw, and cover the rat with an absorbent pad to maintain body temperature. Make a three-to four-centimeter midline skin incision in the neck, and use blunt tip curved scissors to blunt dissect the subcutaneous tissue, to expose the right sternohyoid muscle. Using nontraumatic forceps, move the muscle laterally until the right carotid artery, jugular vein, and vagus nerve are identified.
Use scissors to carefully separate the vagus nerve from the carotid artery. Inject 2, 000 international units per kilogram of heparin into the right jugular vein, applying pressure to the injection site after needle retraction to avoid blood leakage. Use curved forceps to pass two 5-0 silk sutures around the carotid artery.
Firmly attach the distal suture to occlude the carotid artery at the superior aspect of the exposed artery, keeping the proximal suture untied, and place the rat under a stereo microscope. Then use microsurgery scissors to carefully make a one-millimeter incision over the anterior wall of the carotid artery, and insert a 22-gauge, one-inch closed intravenous catheter toward the aortic arch. The use of magnification and microdissection tools is highly recommend for these steps.
A clean field can be achieved by tensioning the proximal suture to avoid bleeding. Cardiac donation is necessary to assemble the ex vivo cardioplegia apparatus. To initiate the DCD protocol, first extubate the animal, then use mosquito forceps to clamp the trachea, starting the agonal phase.
Begin counting the functional WIT when the peak systolic blood pressure drops below 30 millimeters of mercury or asystole or ventricular fibrillation appears, whichever comes first. At the end of WIT, perform a medial sternotomy, keeping the thorax open with an Alm retractor if necessary. Separate the heart from the pulmonary veins and other thoracic structures to complete the cardiectomy.
Immediately submerge the heart in ice-cold Krebs solution for a rapid transportation to the ex vivo system. Use a 2-0 silk suture to tightly fix the aorta to the Langendorff apparatus, and then open the stopcock. Now fully open the flow to the cannula, and start the initial reperfusion and stabilization time.
Rotate the heart so the atria is facing the pressure sensor, and dissect the pulmonary veins to widen the left ventricular atrial opening if necessary. Next, insert a latex balloon connected to a pressure sensor, making sure that the balloon is fully positioned inside the ventricle. Slowly fill the balloon with saline until the end diastolic pressure reaches 15 millimeters of mercury.
Insert the pacing electrode in the anterior face of the heart without puncturing the coronary vessels. Once spontaneous beating is observed, set the pacing to 300 beats per minute. After 10 minutes of stabilization, initiate the continuous intraventricular pressure measurement recording to begin the reconditioning and assessment phase.
After one hour, remove the heart from the Langendorff apparatus and use a straight high-carbon steel blade to remove the atria. With the right ventricle facing down, cut one-to two-millimeter-thick transverse ventricular slices. Excise the right ventricle from the third slice of tissue, and snap freeze the left ventricle for downstream biochemical analyses.
Submerge the remaining sections in freshly prepared 5%TTC solution in PBS for 10 minutes at 37 degrees Celsius. The next morning, wash the samples two times in fresh PBS and submerge the samples in fresh PBS for imaging. Viable tissues will appear brick-red in color.
Following extubation, the blood pressure rapidly drops in a predictable pattern and the expected time to death is less than five minutes. Here, average pressure versus time curves at the start of reconditioning following zero, 10, and 15 minutes of WIT are shown. The contractile function will improve over time, the use of short periods of WIT will allow contractility to return to normal, and morphological damage will not be detectable.
The use of a conditioning agent added with the cardioplegia and at the stabilization phase showed that the damage generated by 15 minutes of WIT in this model are amenable to modulation by cardioprotective agents. This technique allows full control of all the relevant variables and pharmacological and nonpharmacological intervention testing and can be reproduced in larger models, facilitating clinical translation.
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Overview
This protocol targets the limited transplantation organ supply issue by facilitating the evaluation of different cardioprotective conditioning strategies to allow the use of hearts donated after circulatory death. It provides a simple and flexible approach for assessing new conditioning agents or strategies.
Key Study Components
Area of Science
- Cardiac transplantation
- Cardioprotection
- Circulatory death
Background
- Organ supply for transplantation is limited.
- Hearts from donors after circulatory death can be utilized.
- Conditioning strategies may improve heart viability.
- Evaluation of these strategies is crucial for enhancing transplant success.
Purpose of Study
- To evaluate new conditioning agents.
- To increase the feasibility of cardiac donation.
- To improve outcomes for hearts donated after circulatory death.
Methods Used
- Animal model: Anesthetized adult rat.
- Temperature monitoring using a rectal probe.
- Use of a transdermal pulse oximeter for physiological assessment.
- Surgical exposure of the carotid artery and jugular vein for intervention.
Main Results
- Successful identification of key anatomical structures.
- Establishment of a protocol for evaluating cardioprotective strategies.
- Potential for improved heart viability post-transplantation.
- Insights into the effectiveness of conditioning agents.
Conclusions
- This protocol provides a framework for future research.
- It addresses the critical issue of organ supply in transplantation.
- Further studies are needed to validate findings.
Frequently Asked Questions
Executive Industry Relevance
Preclinical models enabling controlled evaluation of cardiac donation after circulatory death (DCD) address a critical bottleneck in transplant medicine by expanding the donor pool. This protocol provides a reproducible system for testing cardioprotective interventions and quantifying ischemia-reperfusion injury, supporting mechanistic de-risking and translational continuity. The approach facilitates risk-adjusted advancement of candidate therapies and conditioning strategies for cardiac transplantation portfolios.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of cardioprotective hypotheses under clinically relevant ischemic conditions.
- Supports mechanistic de-risking by isolating variables such as warm ischemic time and intervention timing.
- Provides functional and structural readouts for target validation of cardioprotective agents.
Screening & Assay Development
- Establishes a standardized ex vivo heart perfusion system for reproducible assessment of candidate interventions.
- Delivers quantitative outputs including intraventricular pressure, troponin T release, and infarct size.
- Facilitates assay scalability and platform reuse for compound screening in cardiac injury models.
Translational & Preclinical Research
- Aligns with disease-relevant endpoints such as ischemia-reperfusion injury and functional recovery.
- Enables continuity from discovery-stage validation to large animal and clinical translation.
- Supports risk-adjusted go/no-go decisions for advancing cardioprotective strategies.
Pipeline & Workflow Integration
This model integrates into the discovery-to-preclinical continuum by enabling hypothesis testing, quantitative screening, and translational validation of cardioprotective interventions for DCD hearts.
- Discovery Biology: Allows precise manipulation of ischemic variables and intervention timing for mechanistic studies.
- Screening: Provides standardized, quantitative functional and biochemical readouts for candidate evaluation.
- Analytics: Supports statistical comparison of intervention efficacy using pressure curves, biomarker release, and infarct quantification.
- Translational Research: Facilitates reproducibility and scalability to larger models, supporting clinical translation.
- Enterprise Reuse: Offers a flexible, reproducible platform for ongoing evaluation of novel cardioprotective agents.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in cardioprotective target validation and reduces mechanistic ambiguity.
- Operational Value: Standardizes assessment of cardiac injury and recovery, improving reproducibility and scalability.
- Strategic Value: Informs go/no-go decisions and capital allocation for cardiac transplantation R&D portfolios.
- Portfolio Impact: Enables risk-adjusted prioritization of candidate interventions for clinical advancement.
Implementation Considerations
- Requires expertise in small animal surgery and ex vivo heart perfusion techniques.
- Needs access to pressure monitoring, perfusion systems, and histological analysis infrastructure.
- Demands rigorous cross-team standardization of ischemic timing and intervention protocols.
- Adaptable to larger animal models for enhanced translational relevance.
- Limitations include species-specific responses and the need for careful control of ischemic variables.
