A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 rates^1,2,3. 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 animals^6,7 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.

Protokół

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

1. 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.
2. Prepare the modified Langendorff perfusion system.
   1. 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.
   2. 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

1. Hardware
   1. 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.
   2. 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.
2. Software
   1. 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).
   2. 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

1. Prepare the animal.
   1. 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.
   2. Transfer the rat onto a small animal surgical platform, place the rat in a supine position, and sterilize the chest with 75% ethanol.
2. Excise the heart.
   1. 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.
   2. Reflect the sternum cranially with tissue forceps to fully expose the heart and lungs.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
3. Suspend the heart.
   1. 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.
   2. 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.
   3. 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.
   4. 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)

1. Equilibrium stage (0-30 min)
   1. 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.
   2. 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.
2. Electrical stimulation stage (30-120 min)
   1. 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.
   2. 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.
3. Rewarming, defibrillation, and beating stage (120-180 min)
   1. After 90 min of VF, use electrodes to give 0.1 J of direct current defibrillation, as shown in Figure 3C.
   2. 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.
   3. 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

1. CK-MB assay
   1. Use an automatic biochemistry analyzer and commercial CK-MB assay kit to determine the level of CK-MB in the collected coronary effusion fluid⁸.
2. Histological analysis
   1. Fix the heart in buffered 10% formalin, dehydrate the heart, and embed it in paraffin.
   2. 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.

Wyniki

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 gr...

Dyskusje

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...

Materiały

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