Using an Isolated Working Rat Heart Model to Test Donor Heart Preservation Strategies

Summary

We describe an ex vivo isolated working rat heart protocol to test donor heart preservation strategies. This paper describes the protocol for use in static cold storage of rodent donor hearts; however, the protocol can also be used for donor hearts obtained after donation after circulatory death and brain death.

Abstract

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, transportation, and transplantation. Previous work from our laboratory showed that the addition of glyceryl trinitrate and erythropoietin to donor heart preservation solutions could significantly improve cardiac functional recovery after prolonged cold storage and in donation after circulatory death hearts. This supplementation protocol has been implemented in clinical use in transplant units around Australia, Belgium, and the United Kingdom. Here, we outline a protocol for testing supplementation strategies using an ex vivo isolated working rat heart (IWRH) perfusion circuit. Using this methodology, supplementation strategies can be tested in the context of prolonged cold static storage, donation after brain death, and donation after circulatory death donor heart preservation. Cardiac functional recovery, measured by aortic flow, coronary flow, cardiac output, pulse pressure, and heart rate, can be used to determine whether a particular preservation strategy can minimize ischemia-reperfusion injury of the donor heart.

Introduction

The success of a heart transplant can be greatly influenced by the preservation method used for the donor heart. Donor hearts are subjected to ischemic insults throughout the organ retrieval, transport, and transplantation process. The degree of ischemic damage to the donor organ can be mitigated by selecting an appropriate donor heart preservation strategy. Cold static storage (CSS) remains the most feasible and common method for donor heart preservation; however, CSS may not necessarily be the best option for all heart donation pathways. For example, hearts procured via the donation after circulatory death (DCD) pathway are maintained and assessed for viability using normothermic machine perfusion, whereas hearts procured via the donation after brain death (DBD) pathway can be preserved using CSS or hypothermic machine perfusion. Another important determinant for the preservation strategy used is the amount of either warm ischemia (for DCD) or cold ischemia (for DBD) the heart is subjected to during organ procurement and transportation.

Many centers use additional pharmacological supplementation of the heart preservation solution to increase ischemic tolerance and improve post-transplant heart function. Rodent studies from our laboratory showed that supplementation of cardioplegia with erythropoietin and glyceryl trinitrate was able to significantly improve cardiac functional recovery after prolonged cold storage of the donor heart^1,2. These studies then progressed to porcine studies of donor heart preservation and provided the pre-clinical groundwork that led to the incorporation of erythropoietin and glyceryl trinitrate into the cardioplegia solution of hearts procured via a DCD pathway utilizing normothermic machine perfusion^3,4,5. Currently, supplementation with erythropoietin and glyceryl trinitrate is also used in Australian transplant units for clinical DBD heart retrievals incorporating CSS. The overall goal of the isolated working rat heart (IWRH) perfusion protocol outlined below is to test donor heart preservation strategies in a laboratory setting and test their efficacy for preserving donor heart function after reperfusion. The isolated working rat heart protocol described has been used for over two decades within our laboratory and has proven extremely useful in screening for potential supplements and/or strategies that can improve donor heart preservation^2,7,8,9.

Protocol

All animals received humane care in compliance with the National Health and Medical Research Council (Australia) guidelines. All animal procedures were approved by the Animal Ethics Committee of the Garvan Institute of Medical Research (Sydney, Australia).

1. Preparation of Krebs-Henseleit (KH) buffer

1. Prepare 2 L of a 20x stock solution of KH buffer containing 2.36 M NaCl, 0.094 M KCl, 0.024 M MgSO₄, 0.024 M KH₂PO₄. Aliquot into 50 mL tubes and store at -20 °C.
2. Prior to the experiment, prepare two 2 L Erlenmyer flasks of 1x KH buffer containing 1900 mL of deionized water, 100 mL of 20x stock KH buffer, 4.20 g of NaHCO₃ (25 mM), and 3.96 g of glucose (11 mM).
   1. Place buffer flasks in a 37 °C water bath and gas the solution with carbogen (95% oxygen, 5% carbon dioxide, flow rate set to 2 L/min) via air stones (slip-on inlet filter) for 5 min, then add 2.8 mL of 1 M CaCl₂ (1.4 mM). Continue bubbling the solution for at least 1 h prior to the start of the experiment. Use of an air stone assists with uniform aeration of the buffer.
   2. Place a small volume (~50 mL) of buffer into a round metal dish at -20 °C to cool and form a layer of iced buffer.

2. Preparation of perfusion circuit

1. Ensure all tubing and glassware are clean with no visible growth/contamination.
2. Set up heights of glass chambers to correspond to required perfusion pressures based on 1 cm H₂O being equivalent to 1.36 mmHg: Langendorff-to-heart 100 cm (approximately 75 mmHg), pre-load working side-to-heart 15 cm (approximately 11 mmHg), heart-to-afterload chamber 100 cm (approximately 75 mmHg).
3. Start circulation of hot water around the circuit at least 10 min before heart perfusion.
4. Place microfiber filters (1.2 µm) into filter holders and secure them tightly.
5. Prime the circuit with KH buffer, ensuring all air is removed, and use a flow control tubing clamp to adjust the flow on the Langendorff side (Figure 1A, B) to ~50 mL/min. The slower flow allows for ease of aortic cannulation and prevents the heart from being dislodged before tying. Place a thermometer within the circuit via a T-shaped tubing connector to monitor the temperature of the buffer at ~37 °C.

3. Setting up of Lab Chart (data analysis) software

1. Ensure Powerlab (data acquisition system) and flowmeter are switched on and connected to the computer prior to opening the data analysis software.
2. Open a new file.
3. Set up data collection by selecting the Settings drop-down menu and selecting Channel Settings. Select for Aortic Pressure, Aortic Flow, and Heart Rate (Figure 2A).
4. Set up each channel with the settings shown in Figure 2B.

4. Preparation of animal for rat heart isolation

NOTE: The methodology for rat heart isolation and perfusion for non-DBD and non-DCD hearts for cold static storage is outlined below. The methods for heart instrumentation onto the perfusion circuit are relatively similar regardless of which donation method is used. The differences in the protocol for DBD and DCD rat heart donation primarily occur from the time the animal anesthesia has taken effect to when the heart is excised from the animal.

1. Weigh the animal, ensuring that its weight is >330 g. The ideal weight range for male rats is 330-420 g.
2. Administer an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Ensure adequate surgical plane of anesthesia based on the absence of a pedal reflex (toe-pinch reflex) and eye blink reflex.
3. Place the animal in a supine position, securing the limbs with tape. Administer oxygen to the animal via a nose cone.
4. Have the KH buffer ice-slush metal container and a small metal container with ice at the dissecting station. Soak a 7.5 cm x 7.5 cm gauze in KH buffer and place it on the container with ice.
5. Once adequate surgical plane of anesthesia is confirmed, shave the skin at the surgical incision site and clean the skin with aseptic such as betadine or 70% alcohol. Make a lateral incision across the abdomen (laparotomy) and move the stomach and intestines to the side to allow access to the left renal vein. Inject 1500 IU heparin and apply pressure to the injection site until the bleeding stops. Allow 1-2 min for heparin to circulate before continuing with heart retrieval.
6. Open the chest cavity by cutting through the rib cage on either side and cutting across the diaphragm. Carefully hold and lift the heart-lung bloc while cutting along the descending aorta, ensuring that the aortic arch remains intact. This will allow for enough aorta length to remain for cannulation.
7. The rat is euthanized by the removal of the heart and lungs while under anesthesia. Submerge the heart-lung bloc in the KH buffer ice slush to allow the heart to arrest.

5. Cannulating the heart onto the isolated working heart perfusion circuit

1. Transfer the heart-lung bloc to a gauze on ice. Dissect excess fat around the aorta, trim the aorta to leave 3-5 mm length for cannulation, and make a small incision ~1-2 mm in the pulmonary artery. This will allow a path for the coronary effluent.
2. Place the trimmed heart-lung bloc (while still on ice) directly underneath the cannulas. Adjust the flow from the Langendorff side using a flow control tubing clamp to facilitate aortic cannulation. Carefully cannulate the aorta onto the left cannula, hold it with a black clamp, and tie it with a 2-0 silk suture.
3. Increase flow from Langendorff perfusion by completely opening the flow control tubing clamp. Open the afterload clamp. Ensure that there is continuous flow from the Langendorff chamber. Start monitoring heart activity with software.
   NOTE: if the aortic flow value on the Flowmeter display is <10 mL/min, ensure that the pulmonary arteriotomy is of sufficient size. A low aortic flow trace/value could also indicate that the aortic cannula has been inserted too far.
4. Adjust the heart-lung bloc so that the anterior surface of the lungs is facing the operator. Tie the left lung lobe with a 2-0 silk suture and cut off excess left lung tissue. Tie the remaining lung lobes in one sweep with a 2-0 silk suture and cut the tissue. Rotate the heart so that the tied lungs are towards the back.
5. Using small Vannas scissors, cut a 2-3 mm edge of the left atrial appendage, ensuring that a small opening into the atria is achieved. Carefully cannulate the left atrial appendage to the left cannula, hold it with a black clamp, and tie it with a 2-0 silk suture. Figure 3A shows the placement of both cannulas.
6. Adjust the heart chamber water jacket so that the heart sits within the center of the chamber.
7. Open the pressure transducer to air and zero the pressure transducer in the software by selecting the Aortic Pressure drop-down menu > Bridge Amp > Zero. Click OK.
8. Perfuse the heart in Langendorff mode for 10 min. Ensure that the Langendoff aortic flow measures between 10-20 mL/min (ideal 20 mL/min).
9. Prior to switching the working mode, ensure that the air stone (slip-on inlet filter) from the Langendorff KH buffer reservoir is placed in the working KH buffer reservoir.
10. Switch the heart into working mode by closing the large clamp leading to the aortic cannula and opening the flow leading to the left atrial appendage. Open three-way tap leading from working perfusate. Transfer the common return tubing (taking KH buffer back to the reservoir) from the Langendorff reservoir to the working reservoir.
11. Perfuse the heart in working mode for 15 min. Aortic flow values must ideally be >30 mL/min at the end of 15 min perfusion. Once the heart has stabilized, start recording data in data analysis software.
12. Measure coronary effluent (coronary flow) at 1 min, 5 min, 10 min, and 15 min.
    NOTE: Coronary flow values should be between 10-20 mL/min. Coronary flow greater than 22 mL/min generally indicates a leak originating from an untied pulmonary vessel. Heart rate should be between 200-300 bpm. Coronary effluent can be collected for downstream analysis of tissue injury markers (e.g., troponin-I, lactate dehydrogenase release).

6. Administering cardioplegia for cold static storage of the heart

1. Fill the cardioplegia chamber (positioned at a height of 60 cm to yield perfusion pressure of approximately 40 mmHg) with 50 mL of designated cardiac preservation solution (with or without supplementation).
2. Lower the water jacket and close the three-way tap on the working mode side.
3. Stop the flow towards the left atrial cannula by closing the clamp.
4. Open the flow to the aortic cannula.
5. Close the clamp from the Langendoff side and on the afterload.
6. Open the clamp from the cardioplegia line and flush 15 mL of cardioplegia out from the pressure line. Close the tap at the pressure line.
7. Allow the heart to be flushed with cardioplegia via the aortic cannula and collect the coronary effluent. Run cardioplegia for 3 min and record the volume of cardioplegia administered.
8. Stop recording data in the software and save the file.
9. After 3 min, close the cardioplegia clamp and the small blue clamp leading to the aortic cannula.
10. Place a bulldog clamp on the aortic cannula and a second bulldog clamp on the left cannula. Disconnect the heart-cannula block from the circuit and place it in a beaker containing cardioplegia in an ice box (Figure 3B, C).
11. Store the heart in the fridge for 6 h (or desired cold storage time).

7. Heart reperfusion after cold static storage

1. Prepare KH buffer as per section 1.
2. Prime the perfusion circuit as per section 2.
3. Open the saved file from baseline perfusion.
4. Carefully remove the heart-cannula block from the ice box and secure it onto the perfusion circuit. Before reconnecting the aortic cannula, use a blunt-end 18 G syringe filled with KH buffer to remove any air from the cannula. Connect the aortic cannula first, open the tubing clamp on the Langendorff side, and ensure the afterload tubing clamp is open.
5. Reconnect the left atrial cannula, ensuring that air has been removed from the cannula before connecting to the perfusion circuit. Keep the working side clamp closed until it is time to switch from Langendorff to working mode.
6. Perfuse the heart in Langendorff mode for 15 min. Ensure to start recording data at the start of Langendorff reperfusion.
7. Prior to switching to the working mode, place both bubbling stones in the KH buffer, open the 3-way tap on the working side, and transfer common return tubing to the working reservoir.
8. Close the white Langendorff clamp, open the white working side clamp, and perfuse the heart for 30 min. Measure and collect coronary effluent at designated time points for downstream analysis (e.g., cardiac injury markers). Ensure to stop-restart software recording to start a new segment (or alternatively, add a comment).
9. At approximately 15 min working mode reperfusion, close the three-way tap.
10. At the end of reperfusion, save the data file, stop the flow through the circuit, and disconnect the heart block. Collect left ventricle samples for histological processing and/or snap frozen for future studies (e.g., protein extraction for western blot studies).
11. Remove the filters from the filter holders, drain the buffer from the circuit, and flush twice with 5 L of distilled water.
    NOTE: Although not necessary, it does help to pre-warm the distilled water in a large container within the water bath.

8. Analysis of functional recovery

1. Open the LabChart file.
2. Highlight a region of at least 30 s of recording, then click Command > Add data selection OR Multiple add to data. This will add the desired parameters to a DataPad file within the software.
3. Select Window drop-down menu > DataPad.
4. Highlight and copy the recovery values from DataPad to a spreadsheet for functional recovery analysis. Calculate functional recovery as a percentage of its respective baseline value^1,6,7.

Results

The results from the baseline perfusion will determine whether the initial experiment (pre-storage) was successful. The aortic flow displayed during Langendorff mode should be between 14 - 22 mL/min. Langendorff flow is displayed as a negative value on the flow meter due to the retrograde perfusion of the aorta. An example of an acceptable Langendorff trace is shown in Figure 4A.

Once switched to working mode, the aortic flow should be at least 30 mL/min once the ...

Discussion

Given the sensitive nature of heart baseline function, care must be taken to maintain the perfusion rig clean and with compatible components. For example, the correct PVC tubing must be used. Some tubing materials, such as silicone, can contribute to lower aortic flow and contractility, which may be due to loss of oxygen via the tubing. While there are isolated working rat heart perfusion circuits commercially available, most of these are primarily used for Langendorff perfusion only. Generally, functional measurements c...

Materials

Name	Company	Catalog Number	Comments
1.5 mL colorless Eppendorff tube, 1000 per box	Bio Strategy Pty Ltd	0030.125.150
15 mL cent/tubes (S) Sleeve/25 PP ctn/500	Sigma-aldrich Pty Ltd	CLS430791-500ea
BP Transducer/Cable kit	ADInstruments	MLT1199	Data Acquisition
Bridge Amp	ADInstruments	FE221	Data Acquisition
Carbogen 555G2	BOC	BOC002
Checktemp1 thermometer	Hanna Instruments	HI98509	Rig Construction
Clamp Pinch 1/4-7/16 PK 12	Thomas Scientific	2848Y40	Rig Construction
Clamp W/Extension Stem Med.	Thomas Scientific	8847T08	Rig Construction
Clamp W/Extension Stem Sm.	Thomas Scientific	8847T02	Rig Construction
Clips, Vessel, 60 g Pressure	Coherent Scientific	14121	Surgical Equipment
Closed Connector	Thomas Scientific	8847E25	Rig Construction
Covidien Sofsilk 2-0 black precut 45 cm box of 24	Specialist Medical Supplies	S195	Surgical consumables
Custom Made 250 mL Jacketed Degasser	Custom Blown Glassware Pty Ltd	N/A	Rig Construction
Custom Made 750 mL Reservoir	Custom Blown Glassware Pty Ltd	N/A	Rig Construction
D-(+)-Glucose Anhydrous SigmaUltra	Sigma-aldrich Pty Ltd	G7528-1Kg	To make Krebs Buffer
Dual Channel Console	ADInstruments	TS402	Data Acquisition
Erlenmyer flasks 2 L
Filter Microfibre type GF/C glass fibre 47 mm, 100	Bio-strategy Pty Ltd	1822047
Forceps, 15 cm, 0.3 mm, CRVD	Coherent Scientific	14114	Surgical Equipment
Four Prong Clamps with 9 mm x 115 mm long arm for holding 2-70 mm diam objects. Vinyl coated	Met-App Australia Pty Ltd	1352	Rig Construction
Heater Circulator. Digital Solid State Control. (1020 Watts/240 Volts)	Thermoline Scientific	TU3	Rig Construction
Heparin 5000 U/5 mL box 50 Pfizer 02112115	Clffird Hallam Healthcare Pty Ltd	1258693	Drugs
Ilium Xylazil 20 Inj 50 mL	Cenvet Australia Pty Ltd	X5010	Anaesthetic
Johns Hopkins Bulldog Clamp	Coherent Scientific	CS-WPI-14117
Ketamine 100 mg/50 mL	Provet (NSW) Pty Ltd	KETAI1	Anaesthetic
Magnesium Sulphate heptahydrate AR 500 g Chemsupply	Bio Strategy Pty Ltd	MA048-500g	To make Krebs Buffer
Male/Female Hinged Adapter	Thomas Scientific	8847V08	Rig Construction
Masterflex L/S Easy-Load Head for Precision Tubing, PPS, CRS Rotor	John Morris	1015164	Rig Construction
Metzenbaum scissors, 11.5 cm curved	Coherent Scientific	WPI-501748	Surgical Equipment
Metzenbaum scissors, 14.5 cm straight	Coherent Scientific	WPI-501252	Surgical Equipment
Mounting Hardware F/2-HEADS SS	John Morris	1014414	Mounting screws for pump heads
Open-sided connector	Thomas Scientific	8847E05	Rig Construction
Paraformadehyde	Sigma-Aldrich	P6148-500G	Sample processing
Potassium Chloride (AnalaR NORMAPUR) 500 g	VWR Chemicals	26764.26	To make Krebs Buffer
Potassium phosphate monobasic	Sigma-Aldrich Pty Ltd	P5379-500g	To make Krebs Buffer
Powerlab 2/26, 2 channel recorder + Labchart software	ADInstruments	ML826	Computer Hardware and Software
Precision XN Inline Flowsensor, 3.2 mm (1/8")ID ME4PXN-KR37 XF	ADInstruments	ME4PXN	Rig Construction
Scalpel with handle disposable #11 pkt/10	BSN Medical (Aust) Pty Ltd	73252-36
Silicone Gasket for Swinnex 47 mm 5/PK	Merck Millipore	SX0004701	Rig Construction
Silicone O-Ring 5-329 10/PK	Merck Millipore	XX1104707	Rig Construction
Single Buret Clamp	Thomas Scientific	8847T32	Rig Construction
Slip-on inlet Filter pore size 10 µm (bubbler)	Sigma-aldrich	59277	Rig Construction
Sm. 360 Rotation Connector	Thomas Scientific	8847E35	Rig Construction
Sodium bicarbonate	Sigma-Aldrich Pty Ltd	S6297 - 250g	To make Krebs Buffer
Swinnex Filter Holder, 47 mm	Merck Millipore	SX0004700	Rig Construction
syringes 1 mL box/100	Becton Dickinson Pty Ltd	302100
Three Prong Clamps with 9 mm diameter x 125 mm long arm and twin screw for holding 5-80 mm	Met-App Australia Pty Ltd	1356	Rig Construction
Tubing Flowmeter Module TS410	ADInstruments	TS410	Data Acquisition
Tubing PVC 6.35 mm ID x 9.52 mm OD 50ft Roll 15.24m, clear , DEHP phthalate free, food grade meets REAC	Thermo Fisher	NAL 8701-0600	Rig Construction
Tubing PVC 7.94mm ID x 11.1mm OD 50ft Roll 15.24 m, clear , DEHP phthalate free, food grade meets REAC	Thermo Fisher	NAL 8701-0900	Rig Construction
Tubing PVC 9.52 mm ID x 12.7 mm OD 100ft Roll	Thermo Fisher	NAL 8701-4120	Rig Construction
Vannas scissors, 8.5 cm, Straight, 7 mm Blades	Coherent Scientific	WPI-500-086	Surgical Equipment
Water Bath 30 Litre with Suspended Tray	Thermoline Scientific	TLWB-30	Rig Construction