This protocol provides a detailed need to develop a long-term ventricular fibrillation rat model using the Langendorff apparatus. It fills the gap of long-term ventricular fibrillation in the small animal model. The technical is simple, economical, repeatable, and stable.
This technique can be used in the study of ventricular fibrillation arrest under perfusion which is performed in certain clinical situations in the field of cardiac surgery sessions and operations. To begin, prepare the Krebs-Henseleit or KH buffer by adding 118 millimolar sodium chloride, 4.7 millimolar potassium chloride, 1.2 millimolar magnesium chloride, 1.2 millimolar sodium dihydrogen phosphate, 1.8 millimolar calcium chloride, 25 millimolar sodium bicarbonate, 11.1 millimolar glucose, and 0.5 millimolar EDTA in distilled water. Then prepare the modified Langendorff perfusion system.
Continuously gas the flask containing KH buffer with 95%oxygen and 5%carbon dioxide at a pressure of approximately 80 millimeters of mercury. Place one end of the perfusion tube in the KH buffer, pass the middle of the perfusion tube through the water bath, and attach a blunt 20 gauge needle to the other end of the perfusion tube. After suspending the needle on a wire stand, adjust the temperature of the water bath so that the temperature of the KH buffer from the end of the perfusion system is 37 degrees Celsius.
Use a physiological signal recorder to digitize and record all the analog signals. Use two stainless steel needle electrodes to record a bipolar electrocardiogram or ECG and 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.
Next, use the laptop software to automatically recognize, adjust, and record bipolar ECG and hemodynamic parameters. Set the electrical stimulator parameters to 30 hertz alternating current with the low voltage group receiving 2 volts and the high voltage group receiving 6 volts. with the anesthetized rat connected to a ventilator after cervical dissection and tracheal intubation, lift the skin off the xiphoid process with toothed forceps and make a three-centimeter transverse incision in the skin with tissue scissors.
Extend the skin and rib incisions to the axillae on both sides in a V shape. 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.
Immediately immerse the heart in a Petri dish with KH buffer at 0 to 4 degrees Celsius to wash and pump out the residual blood. Transfer the heart to a second Petri dish. Cut off the redundant tissue and identify the aorta.
Use two ophthalmic forceps to lift the aorta and insert the aorta into the blunt needle of the Langendorff apparatus. Then with the help of an assistant, tie a knot with a 0 suture thread. Turn on the perfusion flow regulator.
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 to 10 millimeters of mercury. Connect the ECG and electrical stimulation electrodes to the heart.
Place the heart in the center of the jacketed glass chamber maintained at 37 degrees Celsius. Allow the heart to equilibrate for 20 minutes inside the chamber. Then adjust the water bath temperature to maintain the temperature within the jacketed glass chamber at 30 degrees Celsius.
After the temperature has reached the desired level, activate the electrical stimulation switch on the laptop software. If the animal is part of the continuously-stimulated long-term ventricular fibrillation or VF group, allow 90 minutes of electrical stimulation. After 90 minutes of VF, use electrodes to give 0.1 joule of direct current defibrillation.
Simultaneously, regulate the water bath temperature to allow the temperature to rise slowly within the jacketed glass chamber to 37 degrees Celsius for 10 minutes. After defibrillation, allow the heart to beat for 60 minutes and then stop the beating by slow perfusion with 10%potassium chloride at approximately 37 degrees Celsius. Remove the heart for creatine kinase or CK-MB assay and histological analysis.
The rates of VF, the success rate of defibrillation, and the success rate of the VF model are shown here. 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 five minutes had different rates of VF, but the VF rate was slower in both groups compared with Group LC and Group HC.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. The heart rate or HR, coronary flow or CF, and left ventricular pressure difference or LVPD recovery rates are shown here.
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 to long-term VF was poor and the high voltage continuously-stimulated long-term VF group showed the worst recovery rate.
The analysis of coronary effusion fluids 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.This protocol is suitable for study related to ventricular fibrillation arrest and the perfusion in cardiovascular surgery.
