Pre-clinical Model of Cardiac Donation after Circulatory Death

Summary

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.

Abstract

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.

Introduction

Solid organ transplantation in general and cardiac transplantation, in particular, are on the rise worldwide^1,2. 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 pathologies^6,7. For ethical and legal reasons, limited or no cardioprotective interventions can be applied in donors prior to circulatory death^5,8,9. 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 scholars^10,11,12,13. 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 transplantability^6,8.

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.

Protocol

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

1. Turn on the water bath to heat the cardioplegia delivery system (Figure 1A) and the Langendorff ex vivo perfusion system (Figure 1B). Set the water temperature to 38.5 °C for a solution temperature of 37 °C. Setup photographs can be seen in Supplementary Figure 1A,B.
2. Prepare 1 L of cardioplegic solution. Add 1 mL of 2% lidocaine hydrochloride and 10 mL of 2 mM KCl (final concentration 20 mM) to 1 L of Plasma-Lyte A (140 mM Na, 5 mM K, 1.5 mM Mg, 98 mM Cl, 27 mM acetate, 23 mM gluconate). Correct pH to 7.4 using 6 N HCl.
   CAUTION: This model is highly sensitive to pH. A wrong pH correction (outside the 7.3-7.4 physiological range) or pH unstable solutions may compromise the experiment or provide unreliable data.
3. Prepare 4 L of Krebs solution (113 mM NaCl, 4.5 mM KCl, 1.6 mM NaH₂PO₄, 1.25 mM CaCl₂, 1 mM MgCl₂∙6H₂O, 5.5 mM D-Glucose, 25 mM NaHCO₃). Substrate masses per 1 L of solution should be as follows: 6.1 g of NaCl, 0.3355 g of KCl, 0.2035 g of MgCl₂∙6H₂O, 0.192 g of NaH₂PO₄, 0.1387 g of CaCl₂, 0.99 g of D-Glucose, 2.1 g of NaHCO₃, final volume of 1 L in ultrapure deionized water. Add the NaHCO₃ last to avoid precipitation. Filter the solution using a 0.22 µm filter and store overnight. Correct the pH to 7.4 when the solution is at 37 °C and bubble with 5% CO₂/95% O₂.
4. Fill the Langendorff circuit with Krebs solution and start the system pump. Make sure that no bubbles are left inside the tubing. Adjust the peristaltic pump speed to 80 rpm (equivalent to 1 L/min). Using the two way stop cock, adjust the flow to maintain a slow drip through the aortic cannula until the heart is attached (Figure 1B). Keep a sample of Krebs solution (15 mL) in a 50 mL conic tube on ice for heart transportation.
5. Fill the cardioplegia delivery system with the cardioplegic solution. Once the bubbles are removed, switch the circuit to saline using a 3 way stop cock (Figure 1A). Adjust the drip rate. Saline must be slowly dripping from the tip of the catheter to assure that no cardioplegic solution is injected before the animal’s death.

2. Animal Preparation

1. Using an inhalation chamber, induce anesthesia with 3% isoflurane. Once the animal is unresponsive, perform an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (5 mg/kg) or similarly suitable anesthetic, following local regulations, to maintain anesthesia for the rest of the procedure. Ensure the depth of anesthesia by no reaction to toe pinch and palpebral reflex.
2. Intubate the animal using a 14 G, 2-inch I.V. catheter. Start ventilation at 50 breaths per min, with airway pressure limited to 20 cmH₂O.
3. Place the animal on a heating pad set to “medium” and cover with an absorbent pad to maintain body temperature. Insert a rectal temperature probe and attach a transdermal pulse oximeter sensor to one of the feet. Maintain rectal temperature at 37 °C throughout the procedure.
4. Vascular access
   1. Make a 3 to 4 cm midline skin incision in the neck using scissors. Using blunt tip curved scissors, blunt dissect the subcutaneous tissue and expose the right sternohyoid muscle. Using non traumatic forceps, move the muscle laterally until the right carotid artery (pulsating), jugular vein (non-pulsating) and the vagus nerve (white) are visually identified (Supplementary Figure 2A). Carefully separate the vagus nerve from the carotid artery using blunt tip curved scissors.
   2. Inject heparin (2,000 IU/kg) via the right jugular vein. Apply pressure to the injection site after needle retraction to avoid blood leakage.
   3. Using curved forceps, pass two 5-0 silk sutures around the carotid artery. Firmly attach a distal suture to occlude the carotid artery at the superior aspect of the exposed artery. Keep the proximal suture untied. Pulling of the proximal suture will be used for bleeding control in the next step (Supplementary Figure 2B). The distance between sutures should be approximately 2 cm.
   4. Using a stereomicroscope for better visualization, carefully make a 1 mm incision with microsurgery scissors over the anterior wall of the carotid artery. Insert a 22 G, 1-inch closed I.V. catheter towards the aortic arch. The catheter is connected to a 2 way stop cock, allowing for connection to a pressure transducer for constant monitoring, with the possibility of injecting saline or cardioplegia via the cardioplegia delivery system (Figure 1A).

3. Initiation of Cardiac Donation After Circulatory Death (DCD) Protocol

NOTE: A complete protocol timeline can be seen in Figure 2.

1. Re-asses the anesthetic depth by performing a toe pinch and evaluating palpebral reflex. If reaction is observed, perform an intraperitoneal injection of Ketamine (37.5 mg/Kg) and xylazine (2.5 mg/Kg). Re-evaluate after 5 minutes. If no response is observed continue the procedure. Tracheal clamp should only be performed in adequately anesthetized animals.
2. Turn off the ventilator and extubate the animal. Using mosquito forceps, clamp the trachea. This moment is considered as the start of the agonal phase. Start counting the functional warm ischemic time (WIT) when the peak systolic blood pressure drops below 30 mmHg, or if asystole or ventricular fibrillation appears, whatever comes first (Figure 3).
   NOTE: Damage extent should be proportional to WIT. Experiments are needed to optimize WIT time according to anesthetic used, animal strain, sex and weight chosen. In control animals, immediately after carotid vascular access is secured, cardioplegia is injected and the heart is procured as described in the next step (Figure 2). The start of perfusion with cardioplegia is considered as the end of WIT.
3. At the end of WIT, perform a medial sternotomy. Keep the thorax open by using an Alm retractor. Using scissors, open the inferior vena cava and both atria to avoid myocardial distension or cardioplegia recirculation (Supplementary figure 3). Clamp the aorta above the diaphragm. Through the previously catheterized carotid artery, infuse the cardioplegic solution at a constant pressure of 60 mmHg for 5 min using the cardioplegia delivery system. Infusion pressure can be modified by altering the height of the water column.
4. At the end of cardioplegic infusion, dissect the ascending proximal aorta from the pulmonary artery using curved forceps (Supplementary Figure 4A). Cut the aorta distal to the left subclavian artery. Ensure an aortic length of at least 0.5 cm for cannulation for the Langendorff apparatus.
5. Holding the heart from the aorta, complete the cardiectomy by separating the heart from the pulmonary veins and other thoracic structures (Supplementary Figure 4B). Rapidly, submerge the heart in to ice-cold Krebs solution for rapid transportation to the ex vivo system. Keep the dissection and transport times as short as possible (5 min).

4. Ex Vivo Heart Perfusion System (EVHP) and Cardiac Functional Assessment

1. Open the aortic lumen using forceps. Deair the aorta by filling the lumen with the dripping Krebs solution to avoid forcing bubbles in to the coronary vessels. Lower the cannula into the aorta, taking care not to pass the aortic root or damage the aortic valve leaflets. Fix the setup with a small clamp.
2. Using the 2 way stopcock, increase the flow to search for possible leaks in the aorta. If none are detected, tightly fix the aorta to the cannula using a 2-0 silk suture. Fully open the flow to the cannula. Maintain aortic pressure at a physiological pressure of 60-70 mmHg (adjusted by changing the height of the system). At this moment the initial reperfusion and stabilization time is initiated. Aortic pressure can be modified according to the investigator’s experimental plan.
3. Rotate the heart so the base of the heart (atria) is facing the pressure sensor. Widen the left ventricular atrial opening by dissecting the pulmonary veins. Insert the latex balloon connected to a pressure sensor. Make sure that the balloon is fully positioned inside the ventricle by visual inspection. Slowly fill the balloon with saline until end diastolic pressure (EDP) is set to 15 mmHg. Adjust as needed to keep EDP constant (pre-determined physiological EDP). The EDP can be adjusted according to the experimental objectives of each investigator.
4. Insert the pacing electrode in the anterior face of the heart (right ventricular outflow tract). Avoid puncturing the coronary vessels. Once spontaneous beating is observed, initiate pacing at 300 beats per min. Required voltage may vary between experiments and rat strains.
5. After 10 min of stabilization, initiate continuous intraventricular pressure measurement recording. This moment is considered the beginning of the reconditioning and assessment phase (time 0) that will last for 1 h (Figure 2). Reconditioning may be prolonged, but a time-dependent decrease in contractility is expected in all hearts.
6. At the start of reconditioning, collect cardiac effluent dropping from the cardiac veins for 5 min for baseline coronary flow assessment and biochemical analyses. For troponin T repeat every 15 min (times 0, 15, 30, 45 and 60 min). For other analyses individualization of collection times is needed (Figure 2).

5. End of Experience

1. Remove the heart from the Langendorff apparatus.
2. Using a straight high carbon steel blade (microtome blade or similar), remove the base of the heart (including aorta and pulmonary artery).
3. With the right ventricle facing down, cut transverse ventricular slides of 1-2 mm thickness. In one representative section (normally the third) excise the right ventricle and snap freeze the left ventricle. This sample can be used for biochemical analyses.
4. Submerge the remaining sections in to freshly prepared 5% 2,3,5-triphenyl-tetrazolium chloride in commercial phosphate buffer saline pH 7.4 for 10 min at 37 °C. Viable tissues are colored red brick.
5. Wash twice with phosphate buffer saline pH 7.4 and fix with 10% formalin at 4 °C overnight. Wash twice with phosphate buffered saline pH 7.4 and keep each slice submerged.
6. Withdraw excess liquid and weight each slide. Take digital color images of both sides. Use planimetric analyses to calculate percent infarct size and correct for slice and total ventricular weight. Coloration fades with time. Photos must be taken as soon as possible.

6. Data analyses

1. Save all pressure data in a new file per animal.
2. For pressure analyses, select at least 200 pressure cycles per time points. Analyses can be performed off-line (after completion of the experiment) using dedicated software (i.e., LabChart). Common cardiovascular parameters available include: Maximal generated pressure, end diastolic pressure, +dP/dt (steepest slope during the upstroke of the pressure curve, an indicator of ventricular contractile ability), -dP/dt (steepest slope during the downstroke of the pressure curve, an indicator of ventricular relaxation capacity) among others.
   NOTE: For troponin analyses, an increase in troponin release at reperfusion is expected. After 1 h of reperfusion in the EVHP system, troponin levels may decrease to baseline, stressing the need for careful timing in the collection and handling of these samples.

Results

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 (

Discussion

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-cond...

Materials

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride. 1 L bag	Baxter		Electrolyte solution for flushing in the modified Langendorff system.
14 G 2" I.V catheter	Jelco	4098	To act as endotracheal tube.
2,3,5-Triphenyltetrazolium chloride	Milipore-Sigma	T8877	Vital coloration
22 G 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 4x4 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 1,000 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 5 N (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 25 G 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