Summary
This protocol presents a model of long-term ventricular fibrillation in rat hearts induced by continuous stimulation with low-voltage alternating current. This model has a high success rate, is stable, reliable, and reproducible, has a low impact on cardiac function, and causes only mild myocardial injury.
Abstract
Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of intraoperative arrest in the field of cardiac surgery. With recent advances in cardiac surgery, the demand for prolonged VF studies under perfusion has increased. However, the field lacks simple, reliable, and reproducible animal models of chronic ventricular fibrillation. This protocol induces long-term VF through alternating current (AC) electrical stimulation of the epicardium. Different conditions were used to induce VF, including continuous stimulation with a low or high voltage to induce long-term VF and stimulation for 5 min with a low or high voltage to induce spontaneous long-term VF. The success rates of the different conditions, as well as the rates of myocardial injury and recovery of cardiac function, were compared. The results showed that continuous low-voltage stimulation induced long-term VF and that 5 min of low-voltage stimulation induced spontaneous long-term VF with mild myocardial injury and a high rate of recovery of cardiac function. However, the low-voltage, continuously stimulated long-term VF model had a higher success rate. High-voltage stimulation provided a higher rate of VF induction but showed a low defibrillation success rate, poor recovery of cardiac function, and severe myocardial injury. On the basis of these results, continuous low-voltage epicardial AC stimulation is recommended for its high success rate, stability, reliability, reproducibility, low impact on cardiac function, and mild myocardial injury.
Introduction
Cardiac surgery is usually performed via thoracotomy, with blocking of the aorta and perfusion with a cardioplegic solution to arrest the heart. Repeat cardiac surgery can be more challenging than the initial surgery, with higher complication and mortality rates1,2,3. Furthermore, the conventional median sternotomy approach may cause damage to the bridge vessels behind the sternum, the ascending aorta, the right ventricle, and other important structures. Extensive bleeding due to the separation of connective tissue, sternal wound infection, and sternal osteomyelitis due to sternotomy are all possible complications. Extensive dissection increases the risk of lesions and hemorrhage in vital cardiac structures.
With the development of minimally invasive cardiac surgery, incisions have become smaller, and cardiac arrest is sometimes difficult to achieve. Repeat cardiac surgery under ventricular fibrillation (VF)4,5 is safe, feasible, and may provide better myocardial protection. Therefore, this protocol introduces the method of VF cardiac arrest in surgery with minimally invasive extracorporeal circulation. The heart loses effective contraction during VF, and, thus, there is no need to suture and block the ascending aorta during surgery, which simplifies the procedure. However, even if the heart is continuously perfused, long-term VF may still be harmful to the heart.
As this method becomes more widely used, the question of how to protect the heart during VF becomes increasingly relevant. This will require extensive and in-depth studies using animal models of long-term VF. In the past, research in this field has mostly used large animals6,7 and has required cooperation between surgeons, anesthesiologists, perfusionists, and other researchers. These studies took too long, the sample sizes were often small, and the studies generally focused on cardiac function and less on mechanistic and molecular assessments. To date, no study has reported a detailed protocol to establish a long-term VF model.
This protocol, thus, provides the details needed to develop a long-term VF rat model using Langendorff apparatus. The protocol is simple, economical, repeatable, and stable.
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Protocol
All the experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of PLA General Hospital.
1. Preparing the Langendorff apparatus
- Prepare the Krebs-Henseleit (K-H) buffer. To prepare the K-H buffer, add the following to distilled water: 118.0 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 25.0 mM NaHCO3, 11.1 mM glucose, and 0.5 mM EDTA.
- Prepare the modified Langendorff perfusion system.
- Continuously gas the flask containing K-H buffer with 95% O2 + 5% CO2 at a pressure of approximately 80 mmHg. Place one end of the perfusion tube in the K-H buffer, pass the middle of the perfusion tube through the water bath, and attach a blunt 20 G needle to the other end of the perfusion tube.
- Suspend the needle on a wire stand. Adjust the temperature of the water bath so that the temperature of the K-H buffer from the end of the perfusion system is 37.0 °C ± 1.0 °C.
2. Preparing the hardware and software
- Hardware
- Use a physiological signal recorder to digitize and record all the analog signals. Use two stainless steel needle electrodes to record a bipolar electrocardiogram (ECG), and use two stainless steel needle electrodes for electrical stimulation.
- Connect one end of the four electrodes to the physiological signal recorder and the other end close to the area where the heart will be positioned after attachment to the apparatus.
- Software
- Use the laptop software to automatically recognize, adjust, and record the bipolar ECG and hemodynamic parameters. The parameters include the left ventricular pressure difference (LVPD), the difference between the left ventricular developed pressure (LVDP) and the left ventricular end-diastolic pressure (LVEDP), and the heart rate (HR).
- Set the electrical stimulator parameters to 30 Hz AC, with the low voltage group receiving 2 V and the high voltage group receiving 6 V.
3. Preparing the isolated heart
- Prepare the animal.
- Anesthetize Sprague-Dawley (SD) rats with 2% isoflurane after intraperitoneal injections of 0.05 mg/kg buprenorphine and 1,000 IU/kg heparin sodium. Ensure that the rat has stopped responding to toe pinch.
- Transfer the rat onto a small animal surgical platform, place the rat in a supine position, and sterilize the chest with 75% ethanol.
- Excise the heart.
- With the rat connected to a ventilator after cervical dissection and tracheal intubation, lift the skin off the xiphoid process with toothed forceps, and make a 3 cm transverse incision in the skin with tissue scissors. Extend the skin and rib incisions to the axillae on both sides in a V-shape.
- Reflect the sternum cranially with tissue forceps to fully expose the heart and lungs.
- Isolate and bluntly dissect the thymus using two curved forceps. Clamp the thymic tissue, and deflect it laterally on both sides to expose the aorta and its branches.
- Use curved forceps to perform a blunt separation of the aorta and the pulmonary artery, facilitating the later use of ophthalmic scissors to remove the heart and suspend the heart once it has been removed.
NOTE: For those who are new to this procedure, step 3.2.4 can be omitted. - Use blunt dissection to separate the brachiocephalic trunk from the surrounding tissue. Then, clamp the brachiocephalic trunk with curved forceps to facilitate the removal of the heart. Rapidly cut the aorta between the brachiocephalic trunk and the left common carotid artery. The rat dies as soon as the heart is removed.
- Cut off the redundant tissue, and immediately immerse the heart in a Petri dish with K-H buffer at 0-4 °C to wash and pump out the residual blood.
NOTE: The transection of the aorta between the brachiocephalic trunk and the left common carotid artery is recommended because preserving the trunk allows for the identification of the aorta and the estimation of the depth of cannulation.
- Suspend the heart.
- Transfer the heart to a second Petri dish. Identify the aorta. Use two ophthalmic forceps to lift the aorta, and insert the blunt needle into the Langendorff apparatus.
- Adjust the aortic depth to the appropriate position. Have an assistant tie a knot with a 0 suture thread. Then, turn on the perfusion flow regulator.
NOTE: Take care to avoid any air bubbles entering the heart throughout the procedure. Furthermore, be aware that the time from cutting the aorta to the initial perfusion should not exceed 2 min. - Insert a small modified latex balloon connected to a pressure transducer into the left atrium, and push the balloon through the mitral valve into the left ventricle. Fill the balloon with distilled water to achieve an end-diastolic pressure of 5-10 mmHg.
- Connect the ECG and electrical stimulation electrodes to the heart. Then, place the heart in a jacketed glass chamber to maintain an internal temperature of 37.0 °C ± 1.0 °C.
NOTE: Use the following exclusion criteria: heart rate <250 beats per minute; coronary flow (mL/min) <10 mL/min or >25 mL/min. The ECG and electrical stimulation electrode connection positions are shown in Figure 1A, and the jacketed glass chamber is shown in Figure 1B.
4. Perfusing and electrically stimulating the heart (Figure 2)
- Equilibrium stage (0-30 min)
- Start the perfusion, and maintain a temperature of approximately 37 °C until the heart beats spontaneously; then, allow the heart to equilibrate for 20 min.
- Adjust the water bath temperature to maintain the temperature within the jacketed glass chamber at approximately 30 °C.
NOTE: The entire cooling process should last approximately 10 min.
- Electrical stimulation stage (30-120 min)
- After the temperature has reached the desired level, activate the electrical stimulation switch on the laptop software.
NOTE: The bipolar ECG and left ventricular pressure (LVP) at the beginning of electrical stimulation are shown in Figure 3A. - If the animal is part of the continuously stimulated long-term VF group, allow 90 min of electrical stimulation. If the animal is in the induced spontaneous long-term VF group, allow 5 min of electrical stimulation, then turn off the electrical stimulation, and allow 90 min for spontaneous long-term VF, as shown in Figure 3B.
NOTE: For hearts in the spontaneous long-term VF group that do not develop spontaneous VF within 90 min after electrical stimulation, the electrical stimulation is then turned off as they do not meet the inclusion criteria.
- After the temperature has reached the desired level, activate the electrical stimulation switch on the laptop software.
- Rewarming, defibrillation, and beating stage (120-180 min)
- After 90 min of VF, use electrodes to give 0.1 J of direct current defibrillation, as shown in Figure 3C.
- Simultaneously regulate the water bath temperature to allow the temperature to rise slowly within the jacketed glass chamber to about 37 °C. Continue the warming process for approximately 10 min.
- After defibrillation, allow the heart to beat for 60 min, and then stop the beating by slow perfusion with 10% KCl at approximately 37 °C. Remove the heart for further analysis.
NOTE: Hearts that do not beat after defibrillation do not meet the inclusion criteria. In addition, it is important to collect the coronary effusion before cooling (at 20 min), after defibrillation (at 120 min), and at the end of the experiment (at 180 min).
5. Performing the creatine kinase-MB (CK-MB) assay and histological analysis
- CK-MB assay
- Use an automatic biochemistry analyzer and commercial CK-MB assay kit to determine the level of CK-MB in the collected coronary effusion fluid8.
- Histological analysis
- Fix the heart in buffered 10% formalin, dehydrate the heart, and embed it in paraffin.
- Use a microtome to cut the paraffin-embedded tissue into 5 µm sections; then, mount the sections on glass slides, and stain with hematoxylin and eosin9.
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Representative Results
A total of 57 rats were used in the experiments, of which 30 fulfilled the inclusion criteria. The included animals were divided into five groups, with six animals in each group: the control group (Group C), the low-voltage continuously stimulated long-term VF group (Group LC), the high-voltage continuously stimulated long-term VF group (Group HC), the low voltage-induced spontaneous long-term VF group (Group LI), and the high voltage-induced spontaneous long-term VF group (Group HI). The experimental process for each group is shown in Figure 2.
Success rate of the VF models
The rates of VF, the success rate of defibrillation, and the success rate of the VF model are shown in Table 1. Group LC and Group HC received continuous electrical stimulation, and, thus, VF occurred with a 100% success rate, but Group HC demonstrated lower success rates for defibrillation. Group LI and Group HI, in which the electrical stimulation was turned off after 5 min, had different rates of VF, but the VF rate was lower in both groups compared with Group LC and Group HC. While the groups with higher voltages had a higher incidence of VF, this was accompanied by a lower success rate of defibrillation. Both Group LC and Group LI had better defibrillation success rates, but overall, Group LC had the highest model success rate, whereas Group LI had a lower model success rate.
Hemodynamic changes
The HR, coronary flow (CF), and LVPD recovery rates of the five experimental groups are shown in Figure 4A-C. The recovery rate indicates the percentage of the relevant value at the end of the experiment divided by the value at the beginning of the experiment. The hemodynamic data of each group were compared with those of the control group (Group C). The hemodynamics of Group C remained stable during the experiment and showed a slight decrease in HR, CF, and LVPD. The two groups with low voltage-induced VF had similar performance and a good recovery rate. The HR and LVPD were not significantly different in those groups compared with Group C, but the recovery rate of CF was significantly better than in Group C.
In contrast, the hemodynamic recovery rate of the two groups with high voltage-induced long-term VF was poor, and the high-voltage continuously stimulated long-term VF group showed the worst recovery rate.
CK-MB assay and histological analysis results
The CK-MB levels in the coronary effusion fluid reflect myocardial injury. As shown in Figure 4D, the analysis of coronary effusion fluid collected at the end of the experiment showed that CK-MB levels were higher in both high-voltage groups. No differences were found between the two low-voltage groups and Group C. Hematoxylin and eosin staining showed an electrode burn region in Group HC (Figure 5).
| Total number of isolated perfusion hearts | Number of VF | VF rate | Number of beating after defibrillation | Rate of beating after defibrillation | Success rate of VF model | |
| Group C | 6 | - | - | - | - | - |
| Group LC | 7 | 7 | 100% | 6 | 85.71% | 85.71% |
| Group HC | 14 | 14 | 100% | 6 | 42.86% | 42.86% |
| Group LI | 16 | 7 | 43.75% | 6 | 85.71% | 37.50% |
| Group HI | 14 | 10 | 71.43% | 6 | 60.00% | 42.86% |
Table 1: Success rate of the VF model. Abbreviations: VF = ventricular fibrillation; Group C = control group; Group LC = low-voltage continuously stimulated VF group; Group HC = high-voltage continuously stimulated VF group; Group LI = low voltage-induced spontaneous VF group; Group HI = high voltage-induced spontaneous VF group.
Figure 1: Electrode and jacketed glass chamber settings. (A) Position of the electrical stimulation electrodes and bipolar electrocardiogram (ECG) electrodes on an isolated rat heart. The white arrow points to the electrical stimulation electrodes. The black arrow points to the bipolar ECG electrodes. (B) Temperature control with a water bath and jacketed glass chamber during the experiment. Please click here to view a larger version of this figure.
Figure 2: Heart perfusion and electrical stimulation procedure. Abbreviations: a = start cooling; b = start stimulation; c = stop stimulation; d = start rewarming; e = defibrillation. Please click here to view a larger version of this figure.
Figure 3: Bipolar electrocardiogram (ECG) and left ventricular pressure difference (LVPD). (A) Ventricular fibrillation (VF) occurred after starting the alternating current (AC) stimulation. (B) Spontaneous VF occurred after the cessation of AC stimulation. (C) The heart returned to beating after defibrillation. Abbreviations: a = start stimulation; b = stop stimulation; c = defibrillation. Please click here to view a larger version of this figure.
Figure 4: Hemodynamic recovery rate and creatine kinase-MB (CK-MB) values in the coronary effusion fluid collected at the end of the experiment. (A) Heart rate (HR) recovery rates of each group. (B) Coronary flow (CF) recovery rates of each group. (C) Left ventricular pressure difference (LVPD) recovery rates of each group. (D) Creatine kinase-MB (CK-MB) values of each group. Abbreviation: VF = ventricular fibrillation. (A-D) Bars show the mean ± standard deviation (SD). A one-way ANOVA was performed using GraphPad Prism, followed by Tukey's multiple comparisons test. n = 6 rats per group. *: compared with Group C; #: compared with Group LC. P values less than 0.05 were considered statistically significant. */#: P < 0.05; **/##: P < 0.01; ***/###: P < 0.001; ****/####: P < 0.0001. Please click here to view a larger version of this figure.
Figure 5: Hematoxylin and eosin staining of myocardial tissue at the apex. The green square is the electric stimulation electrode burn region of Group HC. Abbreviation: Group HC = high-voltage continuously stimulated long-term ventricular fibrillation group. Please click here to view a larger version of this figure.
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Discussion
This protocol establishes an animal model of long-term VF in isolated rat hearts that has not been previously reported. Additionally, different electrical stimulation conditions were compared in this study. This study provides a model for studies related to ventricular fibrillation arrest during cardiac surgery.
The success rate of the model is a very important indicator that is related to personnel, time, and economic costs. In VF models, the success rate includes whether VF can be induced in the heart and whether the heart can return to normal beating after defibrillation. In addition, the heart function recovery rate and myocardial injury should be considered. To be an appropriate model for cardiac surgery requirements, the VF time of the heart needs to reach 1-2 h at low temperatures, and, thus, in this protocol, the VF time is 90 min.
Using a low voltage is suggested to have little effect on cardiac function and myocardial injury. Therefore, this study compared the success rates of using low and high voltages, as well as the success rates of continuous or 5 min electrical stimulation to induce VF in rat hearts. Six eligible VF models were made for each group. A total of 16 rats were tested in Group LI, with a model success rate of 37.50%, while only 7 rats were tested in Group LC, with a success rate of 85.71%. Furthermore, in this study, there were no significant differences in the HR, LVPD recovery rate, or CK-MB levels between Group LC and Group LI.
A sufficient intensity of electrical stimulation during the vulnerable period of the cardiac cycle produces VF10. In this study, Group HC and Group HI had a higher incidence of VF than the other groups. However, the CK-MB analysis and hematoxylin and eosin staining results suggested that the high-voltage stimulation could cause significant myocardial damage, leading to a low defibrillation rate. Furthermore, the defibrillation rate of the heart after VF was significantly lower in the high-voltage groups than in the low-voltage groups.
These data show that the low-voltage continuously stimulated long-term VF was the best model with the highest model success rate, a good cardiac function recovery rate after defibrillation, and less myocardial injury.
The rate of CF recovery was better in the two low-voltage groups than in Group C, consistent with reports of similar studies. In a previous study, canine hearts under cardiopulmonary bypass (CPB) showed a significant increase in flow via dilated coronary arteries11, which increased the subendocardial flow three times higher than the epicardial flow. This increased coronary flow may provide sufficient oxygen to meet the increased metabolic demand. Therefore, in the canine model, the normal ventricle shows no metabolic or functional impairment or histological changes after 30-60 min of spontaneous VF. In another CPB canine heart study12, the CF was higher in both spontaneously and continuously stimulated VF than in normal empty beating hearts.
To simulate the temperature during cardiac surgery, the temperature of the K-H buffer and the ambient temperature were controlled at approximately 30 °C during VF in this study. The left ventricular distensibility decreased with hypothermia in beating hearts but increased with hypothermia in VF hearts. In a previous study, the myocardial oxygen consumption in VF hearts was higher than in normal empty beating hearts at 37 °C and lower than in the empty beating hearts at 28 °C13. Therefore, lowering the temperature has more benefits in the perfused VF heart.
The position of the electrodes can affect the occurrence of VF. In this protocol, the needle electrodes are anchored at the base and apex of the right ventricle to obtain electrical stimulation throughout the heart and obtain coronary effusion fluid for biochemical analysis. A previous study anchored an electrode on the endocardium of the right ventricle, placed the other pole in the K-H buffer, and immersed the heart in the K-H buffer14. In addition, studies have reported octapolar electrophysiology catheter placement in the right ventricular endocardium15, epicardial multi-site photostimulation16, and epicardial electrical stimulation with an epicardial multi-electrode array (MEA)17.
In a previous report, researchers performed 3 min of electrical stimulation of the isolated rat heart at 37 °C with 0.05 mA 30 Hz AC to obtain 20 min of VF without perfusion14. A 10-30 Hz AC has also been used to induce VF in isolated nonischemic ferret hearts18. In addition, 1.5-4.5 V AC12, 7.5 V AC13, and unrestricted-voltage AC19 have been used in cardiopulmonary bypass experiments in dogs. Notably, the voltage or current thresholds for induced VF differ between isolated and in vivo hearts, with smaller stimulus intensities in isolated hearts20. In various studies in which VF has been induced with AC, the primary factor influencing the results was the intensity rather than the frequency of electrical stimulation. The frequency of electrical stimulation was not the same in any of these studies, but it has also been noted that 30 Hz produces a higher incidence of VF than 10 Hz21. Direct current (DC) has also been used in studies of electrically stimulated VF, but DC is more commonly used in short-term VF because the threshold for DC to induce VF is three times higher than that for AC22. Moreover, DC may aggravate myocardial injury under prolonged stimulation at high energy. Electrical defibrillation can also cause myocardial injury, but studies have shown significant injury only with much higher defibrillation energy than used in this protocol23.
Attention to a number of steps is essential to make this protocol successful. The ventilator must be connected after anesthetizing the rats to avoid ischemia caused by respiratory arrest, which can confound the experimental results. After removing the heart, it should be immersed, especially the aortic root, in a 0-4 °C K-H buffer, and the heart should be suspended rapidly before it contracts to avoid air entering the heart. The needle must not enter too deeply into the aorta, as this can reduce the coronary perfusion. When suspending the heart, the silk ligation of the aortic root should include the brachiocephalic trunk; otherwise, the coronary perfusion will be shunted. An abnormal increase in coronary flow helps to identify this issue. The depth of the electrode should be approximately 1 mm; electrodes placed too deep will penetrate the ventricular wall, and those placed too shallow may be dislodged.
For certain studies, investigators may wish to simulate a state of long-term spontaneous VF, but the small mammalian heart is characterized by a high rate of spontaneous defibrillation24,25. The long refractory period, rapid conduction, and small mass are not conducive to the maintenance of VF, and the heart returns to a normal rhythm within a short time. Similar studies have been previously performed on small animals with different conditions of VF; however, these studies all assessed short-duration VF. Spontaneous VF does not occur by cooling alone and must be induced by electrical stimulation under different conditions, which is a limitation of this study.
In short, low-voltage continuous epicardial AC stimulation showed a high success rate, stability, reliability, and reproducibility, especially since it had the characteristics of a low impact on cardiac function and low myocardial injury, making this a scalable model of prolonged VF.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
This work was carried out with the support of Cardiovascular Surgery, First Medical Center, Chinese PLA General Hospital and the Laboratory Animal Center, Chinese PLA General Hospital.
Materials
| Name | Company | Catalog Number | Comments |
| 0 Non-absorbable suture | Ethicon, Inc. | Preparation of the isolated heart | |
| 95% O2 + 5% CO2 | Beijing BeiYang United Gas Co., Ltd. | K-H buffer | |
| AcqKnowledge software | BIOPAC Systems Inc. | Version 4.2.1 | Software |
| Automatic biochemistry analyzer | Rayto Life and Analytical Sciences Co., Ltd. | Chemray 800 | CK-MB assay |
| BIOPAC research systems | BIOPAC Systems Inc. | MP150 | Hardware |
| Blunt needle (20 G, TWLB) | Tianjin Hanaco MEDICAL Co., Ltd. | H-113AP-S | Modified Langendorff perfusion system |
| Calcium chloride | Sinopharm Chemical Reagent Co.,Ltd | 10005861 | K-H buffer |
| CK-MB assay kits | Changchun Huili Biotech Co., Ltd. | C060 | CK-MB assay |
| Curved forcep | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| EDTA | Sinopharm Chemical Reagent Co.,Ltd | 10009717 | K-H buffer |
| Electrical stimulator | BIOPAC Systems Inc. | STEMISOC | Hardware |
| Filter | Tianjin Hanaco MEDICAL Co., Ltd. | H-113AP-S | |
| Glucose | Sinopharm Chemical Reagent Co.,Ltd | 63005518 | K-H buffer |
| Heparin sodium | Tianjin Biochem Pharmaceutical Co., Ltd. | H120200505 | Preparation of the isolated heart |
| Isoflurane | RWD Life Science Co.,LTD | 21082201 | Preparation of the isolated heart |
| Magnesium sulfate | Sinopharm Chemical Reagent Co.,Ltd | 20025118 | K-H buffer |
| Needle electrodes | BIOPAC Systems Inc. | EL452 | Hardware |
| Ophthalmic clamp | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Ophthalmic forceps | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Ophthalmic scissors | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Perfusion tube | Tianjin Hanaco MEDICAL Co., Ltd. | H-113AP-S | Modified Langendorff perfusion system |
| Potassium chloride | Sinopharm Chemical Reagent Co.,Ltd | 10016318 | K-H buffer |
| Sodium bicarbonate | Sinopharm Chemical Reagent Co.,Ltd | 10018960 | K-H buffer |
| Sodium chloride | Sinopharm Chemical Reagent Co.,Ltd | 10019318 | K-H buffer |
| Sodium dihydrogen phosphate dihydrate | Sinopharm Chemical Reagent Co.,Ltd | 20040718 | K-H buffer |
| Sprague-Dawley (SD) rats | SPF (Beijing) biotechnology Co., Ltd. | Male, 300-350g | Preparation of the isolated heart |
| Thermometer | Jiangsu Jingchuang Electronics Co., Ltd. | GSP-6 | Modified Langendorff perfusion system |
| Tissueforceps | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Tissue scissors | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Toothed forceps | Shanghai Medical Instrument (Group) Co., Ltd. | Preparation of the isolated heart | |
| Ventilator | Chengdu Instrument Factory | DKX-150 | Preparation of the isolated heart |
| Water bath1 | Ningbo Scientz Biotechnology Co.,Ltd. | SC-15 | Modified Langendorff perfusion system |
| Water bath2 | Shanghai Yiheng Technology Instrument Co., Ltd. | DK-8D | Modified Langendorff perfusion system |
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