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.

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
<ol>
	<li>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, an...

<ol>
	<li>Kilic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcomes+of+mitral+valve+reoperations+in+the+United+States:+An+analysis+of+the+society+of+thoracic+surgeons+national+database.">Clinical outcomes of mitral valve reoperations in the United States: An analysis of the society of thoracic surgeons national database.</a> The Annals of Thoracic Surgery. 107 (3), 754-759 (2019).</li><li>Akins, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+reoperative+valve+replacement+for+failed+mitral+and+aortic+bioprostheses.">Risk of reoperative valve replacement for failed mitral and aortic bioprostheses.</a> The Annals of Thoracic Surgery. 65 (6), 1551-1542 (1998).</li><li>Jamieson, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reoperation+for+bioprosthetic+mitral+structural+failure:+risk+assessment.">Reoperation for bioprosthetic mitral structural failure: risk assessment.</a> Circulation. 108 (Suppl 1), 98 (2003).</li><li>Seeburger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+mitral+valve+surgery+after+previous+sternotomy:+Experience+in+181+patients.">Minimally invasive mitral valve surgery after previous sternotomy: Experience in 181 patients.</a> The Annals of Thoracic Surgery. 87 (3), 709-714 (2009).</li><li>Arcidi, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fifteen-year+experience+with+minimally+invasive+approach+for+reoperations+involving+the+mitral+valve.">Fifteen-year experience with minimally invasive approach for reoperations involving the mitral valve.</a> The Journal of Thoracic and Cardiovascular Surgery. 143 (5), 1062-1068 (2012).</li><li>Cox, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+safety+of+induced+ventricular+fibrillation+during+cardiopulmonary+bypass+in+nonhypertrophied+hearts.">The safety of induced ventricular fibrillation during cardiopulmonary bypass in nonhypertrophied hearts.</a> The Journal of Thoracic and Cardiovascular Surgery. 74 (3), 423-432 (1977).</li><li>Schraut, W., Lamberti, J. J., Kampman, K., Glagov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+fibrillation+during+cardiopulmonary+bypass:+Long-term+effects+on+myocardial+morphology+and+function.">Ventricular fibrillation during cardiopulmonary bypass: Long-term effects on myocardial morphology and function.</a> The Annals of Thoracic Surgery. 27 (3), 230-234 (1979).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pravastatin+attenuates+cardiac+dysfunction+induced+by+lysophosphatidylcholine+in+isolated+rat+hearts.">Pravastatin attenuates cardiac dysfunction induced by lysophosphatidylcholine in isolated rat hearts.</a> European Journal of Pharmacology. 640 (1-3), 139-142 (2010).</li><li>Lang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL10/IP-10+neutralization+can+ameliorate+lipopolysaccharide-induced+acute+respiratory+distress+syndrome+in+rats.">CXCL10/IP-10 neutralization can ameliorate lipopolysaccharide-induced acute respiratory distress syndrome in rats.</a> PLoS One. 12 (1), e0169100 (2017).</li><li>Lubbe, W. F., Bricknell, O. L., Marzagao, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+fibrillation+threshold+and+vulnerable+period+in+the+isolated+perfused+rat+heart.">Ventricular fibrillation threshold and vulnerable period in the isolated perfused rat heart.</a> Cardiovascular Research. 9 (5), 613-620 (1975).</li><li>Hottentrott, C. E., Towers, B., Kurkji, H. J., Maloney, J. V., Buckberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hazard+of+ventricular+fibrillation+in+hypertrophied+ventricles+during+cardiopulmonary+bypass.">The hazard of ventricular fibrillation in hypertrophied ventricles during cardiopulmonary bypass.</a> The Journal of Thoracic and Cardiovascular Surgery. 66 (5), 742-753 (1973).</li><li>Hottenrott, C., Maloney, J. V., Buckberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+effects+of+ventricular+fibrillation+on+the+adequacy+of+regional+myocardial+flow.+I.+Electrical+vs.+spontaneous+fibrillation.">Studies of the effects of ventricular fibrillation on the adequacy of regional myocardial flow. I. Electrical vs. spontaneous fibrillation.</a> The Journal of Thoracic and Cardiovascular Surgery. 68 (4), 615-625 (1974).</li><li>Buckberg, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+effects+of+hypothermia+on+regional+myocardial+blood+flow+and+metabolism+during+cardiopulmonary+bypass.+I.+The+adequately+perfused+beating,+fibrillating,+and+arrested+heart.">Studies of the effects of hypothermia on regional myocardial blood flow and metabolism during cardiopulmonary bypass. I. The adequately perfused beating, fibrillating, and arrested heart.</a> The Journal of Thoracic and Cardiovascular Surgery. 73 (1), 87-94 (1977).</li><li>Gazmuri, R. J., Berkowitz, M., Cajigas, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+effects+of+ventricular+fibrillation+in+the+isolated+rat+heart.">Myocardial effects of ventricular fibrillation in the isolated rat heart.</a> Critical Care Medicine. 27 (8), 1542-1550 (1999).</li><li>Clasen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+approach+for+programmed+electrical+stimulation+in+mice:+Inducibility+of+ventricular+arrhythmias.">A modified approach for programmed electrical stimulation in mice: Inducibility of ventricular arrhythmias.</a> PLoS One. 13 (8), e0201910 (2018).</li><li>Diaz-Maue, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+cardiac+rhythm+management+by+applying+optogenetic+multi-site+photostimulation+in+murine+hearts.">Advanced cardiac rhythm management by applying optogenetic multi-site photostimulation in murine hearts.</a> Journal of Visualized Experiments. (174), e62335 (2021).</li><li>Jungen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+intracardiac+neurons+on+cardiac+electrophysiology+and+arrhythmogenesis+in+an+ex+vivo+Langendorff+system.">Impact of intracardiac neurons on cardiac electrophysiology and arrhythmogenesis in an ex vivo Langendorff system.</a> Journal of Visualized Experiments. 135, e57617 (2018).</li><li>Koretsune, Y., Marban, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+calcium+in+the+pathophysiology+of+ventricular+fibrillation+and+in+the+pathogenesis+of+postarrhythmic+contractile+dysfunction.">Cell calcium in the pathophysiology of ventricular fibrillation and in the pathogenesis of postarrhythmic contractile dysfunction.</a> Circulation. 80 (2), 369-379 (1989).</li><li>Brazier, J. R., Cooper, N., McConnell, D. H., Buckberg, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+effects+of+hypothermia+on+regional+myocardial+blood+flow+and+metabolism+during+cardiopulmonary+bypass.+III.+Effects+of+temperature,+time,+and+perfusion+pressure+in+fibrillating+hearts.">Studies of the effects of hypothermia on regional myocardial blood flow and metabolism during cardiopulmonary bypass. III. Effects of temperature, time, and perfusion pressure in fibrillating hearts.</a> The Journal of Thoracic and Cardiovascular Surgery. 73 (1), 102-109 (1977).</li><li>von Planta, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiopulmonary+resuscitation+in+the+rat.">Cardiopulmonary resuscitation in the rat.</a> Journal of Applied Physiology. 65 (6), 2641-2647 (1988).</li><li>Luo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ageing+increases+cardiac+electrical+remodelling+in+rats+and+mice+via+NOX4/ROS/CaMKII-mediated+calcium+signalling.">Ageing increases cardiac electrical remodelling in rats and mice via NOX4/ROS/CaMKII-mediated calcium signalling.</a> Oxidative Medicine and Cellular Longevity. 2022, 8538296 (2022).</li><li>Hohnloser, S., Weirich, J., Antoni, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+direct+current+on+the+electrical+activity+of+the+heart+and+on+its+susceptibility+to+ventricular+fibrillation.">Influence of direct current on the electrical activity of the heart and on its susceptibility to ventricular fibrillation.</a> Basic Research in Cardiology. 77 (3), 237-249 (1982).</li><li>Xie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-energy+defibrillation+increases+the+severity+of+postresuscitation+myocardial+dysfunction.">High-energy defibrillation increases the severity of postresuscitation myocardial dysfunction.</a> Circulation. 96 (2), 683-688 (1997).</li><li>Manoach, M., Netz, H., Erez, M., Weinstock, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+self-defibrillation+in+mammals:+Age+and+drug+dependence.">Ventricular self-defibrillation in mammals: Age and drug dependence.</a> Age and Ageing. 9 (2), 112-116 (1980).</li><li>Filippi, S., Gizzi, A., Cherubini, C., Luther, S., Fenton, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+hypothermic+ventricular+fibrillation:+The+role+of+temperature+and+tissue+size.">Mechanistic insights into hypothermic ventricular fibrillation: The role of temperature and tissue size.</a> Europace. 16 (3), 424-434 (2014).</li></ol>

The authors have nothing to disclose.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0 Non-absorbable suture</td>
			<td>Ethicon, Inc.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>95% O2 + 5% CO2</td>
			<td>Beijing BeiYang United Gas Co., Ltd.&#160;</td>
			<td></td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>AcqKnowledge software</td>
			<td>BIOPAC Systems Inc.</td>
			<td>Version 4.2.1</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Automatic biochemistry analyzer</td>
			<td>Rayto Life and Analytical Sciences Co., Ltd.</td>
			<td>Chemray 800</td>
			<td>CK-MB assay</td>
		</tr>
		<tr>
			<td>BIOPAC research systems</td>
			<td>BIOPAC Systems Inc.</td>
			<td>MP150</td>
			<td>Hardware</td>
		</tr>
		<tr>
			<td>Blunt needle (20 G, TWLB)</td>
			<td>Tianjin Hanaco MEDICAL Co., Ltd.</td>
			<td>H-113AP-S</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10005861</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>CK-MB assay kits&#160;</td>
			<td>Changchun Huili Biotech Co., Ltd.</td>
			<td>C060</td>
			<td>CK-MB assay</td>
		</tr>
		<tr>
			<td>Curved forcep</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10009717</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Electrical stimulator</td>
			<td>BIOPAC Systems Inc.</td>
			<td>STEMISOC</td>
			<td>Hardware</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Tianjin Hanaco MEDICAL Co., Ltd.</td>
			<td>H-113AP-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>63005518</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Heparin sodium</td>
			<td>Tianjin Biochem Pharmaceutical Co., Ltd.</td>
			<td>H120200505</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co.,LTD</td>
			<td>21082201</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>20025118</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Needle electrodes</td>
			<td>BIOPAC Systems Inc.</td>
			<td>EL452</td>
			<td>Hardware</td>
		</tr>
		<tr>
			<td>Ophthalmic clamp</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Ophthalmic forceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Ophthalmic scissors</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Perfusion tube</td>
			<td>Tianjin Hanaco MEDICAL Co., Ltd.</td>
			<td>H-113AP-S</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10016318</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10018960</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10019318</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate dihydrate</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>20040718</td>
			<td>K-H buffer</td>
		</tr>
		<tr>
			<td>Sprague-Dawley (SD) rats</td>
			<td>SPF (Beijing) biotechnology Co., Ltd.</td>
			<td>Male, 300-350g</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>Jiangsu Jingchuang Electronics Co., Ltd.</td>
			<td>GSP-6</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Tissueforceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Toothed forceps</td>
			<td>Shanghai Medical Instrument (Group) Co., Ltd.</td>
			<td></td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>Chengdu Instrument Factory</td>
			<td>DKX-150</td>
			<td>Preparation of the isolated heart</td>
		</tr>
		<tr>
			<td>Water bath1</td>
			<td>Ningbo Scientz Biotechnology Co.,Ltd.</td>
			<td>SC-15</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
		<tr>
			<td>Water bath2</td>
			<td>Shanghai Yiheng Technology Instrument Co., Ltd.</td>
			<td>DK-8D</td>
			<td>Modified Langendorff perfusion system</td>
		</tr>
	</tbody>
</table>

a model of long-term ventricular fibrillation in isolated rat hearts

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.

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...

Watch this Scientific Journal Video about A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts at JoVE.com

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

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.

Ventricular fibrillation (VF) is a fatal arrhythmia with a high incidence in cardiac patients, but VF arrest under perfusion is a neglected method of ...

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.

a-model-of-long-term-ventricular-fibrillation-in-isolated-rat-hearts

Research

JoVE Journal

Medicine

773 ビュー.  Chinese PLA General Hospital. このプロトコルは、ランゲンドルフ装置を用いて長期心室細動ラットモデルを開発するための詳細な必要性を提供する。小動物モデルにおける長期心室細動のギャップを埋めます。技術はシンプルで、経済的で、再現性があり、安定しています。この技術は、心臓手術セッションおよび手術の分野で特定の臨床状況で行われる灌流下での心室細動停止の研究に使用?...