The original study in which this protocol was used was supported by National Heart, Lung, and Blood Institute Grants HL-103642 and HL-088206

This investigation was conducted according to the National Institutes of Health&#39;s guidelines on animal care and use and was approved by the Boston Children&#39;s Hospital&#39;s Animal Care and Use Committee (Protocol 20-08-4247R). All the animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.
1. Animal species, anesthetic, and analgesic agents
<ol>
	<li>Animal species: Use New Zealand White rabbits (wild type strain; female ...

<ol>
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N., 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=Report+of+the+National+Heart,+Lung,+and+Blood+Institute+Special+Emphasis+Panel+on+Heart+Failure+Research.">Report of the National Heart, Lung, and Blood Institute Special Emphasis Panel on Heart Failure Research.</a> Circulation. 95 (4), 766-770 (1997).</li><li>Rousou, A. J., Ericsson, M., Federman, M., Levitsky, S., McCully, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opening+of+mitochondrial+KATP+channels+enhances+cardioprotection+through+the+modulation+of+mitochondrial+matrix+volume,+calcium+accumulation,+and+respiration.">Opening of mitochondrial KATP channels enhances cardioprotection through the modulation of mitochondrial matrix volume, calcium accumulation, and respiration.</a> American Journal of Physiology. Heart and Circulatory Physiology. 287 (5), H1967-H1976 (2004).</li><li>Rao, V., 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=Insulin+cardioplegia+for+elective+coronary+bypass+surgery.">Insulin cardioplegia for elective coronary bypass surgery.</a> The Journal of Thoracic and Cardiovascular Surgery. 119 (6), 1176-1184 (2000).</li><li>Vinten-Johansen, J., Zhao, Z. Q., Jiang, R., Zatta, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+protection+in+reperfusion+with+postconditioning.">Myocardial protection in reperfusion with postconditioning.</a> Expert Review of Cardiovascular Therapy. 3 (6), 1035-1045 (2005).</li><li>Wakiyama, H., 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=Selective+opening+of+mitochondrial+ATP-sensitive+potassium+channels+during+surgically+induced+myocardial+ischemia+decreases+necrosis+and+apoptosis.">Selective opening of mitochondrial ATP-sensitive potassium channels during surgically induced myocardial ischemia decreases necrosis and apoptosis.</a> European Journal of Cardio-Thoracic Surgery. 21 (3), 424-433 (2002).</li><li>Chen, Q., Moghaddas, S., Hoppel, C. L., Lesnefsky, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+blockade+of+electron+transport+during+ischemia+protects+mitochondria+and+decreases+myocardial+injury+following+reperfusion.">Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion.</a> Journal of Pharmacology and Experimental Therapeutics. 319 (3), 1405-1412 (2006).</li><li>Lesnefsky, E. 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=rather+than+reperfusion,+inhibits+respiration+through+cytochrome+oxidase+in+the+isolated,+perfused+rabbit+heart:+Role+of+cardiolipin.">rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: Role of cardiolipin.</a> American Journal of Physiology. Heart and Circulatory Physiology. 287 (1), H258-H267 (2004).</li><li>McCully, J. D., Rousou, A. J., Parker, R. A., Levitsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-+and+gender-related+differences+in+mitochondrial+oxygen+consumption+and+calcium+with+cardioplegia+and+diazoxide.">Age- and gender-related differences in mitochondrial oxygen consumption and calcium with cardioplegia and diazoxide.</a> The Annals of Thoracic Surgery. 83 (3), 1102-1109 (2007).</li><li>Mandel, I. 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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=Remote+ischemic+preconditioning+reduces+myocardial+and+renal+injury+after+elective+abdominal+aortic+aneurysm+repair:+A+randomized+controlled+trial.">Remote ischemic preconditioning reduces myocardial and renal injury after elective abdominal aortic aneurysm repair: A randomized controlled trial.</a> Circulation. 116, 98-105 (2007).</li><li>Pogwizd, S. M., Bers, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+models+of+heart+disease.">Rabbit models of heart disease.</a> Drug Discovery Today: Disease Models. 5 (3), 185-193 (2008).</li><li>Tanaka, K., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reperfusion-induced+arrhythmias+in+the+isolated+rabbit+heart:+characterization+of+the+influence+of+the+duration+of+regional+ischemia+and+the+extracellular+potassium+concentration.">Reperfusion-induced arrhythmias in the isolated rabbit heart: characterization of the influence of the duration of regional ischemia and the extracellular potassium concentration.</a> Journal of Molecular and Cellular Cardiology. 20 (3), 201-211 (1988).</li><li>Milani-Nejad, N., Janssen, P. M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+and+large+animal+models+in+cardiac+contraction+research:+advantages+and+disadvantages.">Small and large animal models in cardiac contraction research: advantages and disadvantages.</a> Pharmacology &amp; Therapeutics. 141 (3), 235-249 (2014).</li><li>Gupta, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+cardiac+myosin-isoform+shift+during+hypertrophy+and+heart+failure.">Factors controlling cardiac myosin-isoform shift during hypertrophy and heart failure.</a> Journal of Molecular and Cellular Cardiology. 43 (4), 388-403 (2007).</li><li>Lapierre, H., Chan, V., Sohmer, B., Mesana, T. G., Ruel, M. <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+coronary+artery+bypass+grafting+via+a+small+thoracotomy+versus+off-pump:+A+case-matched+study.">Minimally invasive coronary artery bypass grafting via a small thoracotomy versus off-pump: A case-matched study.</a> European Journal of Cardio-Thoracic Surgery. 40 (4), 804-810 (2011).</li><li>Aubin, H., Akhyari, P., Lichtenberg, A., Albert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additional+right-sided+upper+&amp;#34;half-mini-thoracotomy&amp;#34;+for+aortocoronary+bypass+grafting+during+minimally+invasive+multivessel+revascularization.">Additional right-sided upper &amp;#34;half-mini-thoracotomy&amp;#34; for aortocoronary bypass grafting during minimally invasive multivessel revascularization.</a> Journal of Cardiothoracic Surgery. 10, 130 (2015).</li><li>Hu, N., 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=Ligation+of+the+left+circumflex+coronary+artery+with+subsequent+MRI+and+histopathology+in+rabbits.">Ligation of the left circumflex coronary artery with subsequent MRI and histopathology in rabbits.</a> Journal of the American Association for Laboratory Animal Science. 49 (6), 838-844 (2010).</li><li>Sievers, R. 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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=Quantification+of+area+at+risk+during+coronary+occlusion+and+degree+of+myocardial+salvage+after+reperfusion+with+technetium-99m+methoxyisobutyl+isonitrile.">Quantification of area at risk during coronary occlusion and degree of myocardial salvage after reperfusion with technetium-99m methoxyisobutyl isonitrile.</a> Circulation. 82 (4), 1424-1437 (1990).</li><li>Masuzawa, 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=Transplantation+of+autologously+derived+mitochondria+protects+the+heart+from+ischemia-reperfusion+injury.">Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury.</a> American Journal of Physiology. 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Our protocol demonstrates a reliable methodology for performing acute regional myocardial ischemia in the rabbit. The left mini-thoracotomy approach is ideal for survival cases, for which the incision and associated pain must be minimized. Importantly, diuretic therapy was not necessary prior to extubation, and there was no mortality intraoperatively in the non-survival group or at 4 weeks postoperatively in the survival group. When the design of the protocol requires a non-survival case, or when more detailed monitoring...

Cardiovascular diseases are the leading cause of death in the world and contribute to over 18 million deaths each year1,2,3. Acute myocardial infarction (MI) is a common medical emergency that develops when a blood clot or a piece of atheromatous plaque blocks the blood flow of a coronary artery. This causes regional myocardial ischemia in the territory that the artery perfuses.
The present study describes a protocol that utilizes a simple and reliable methodology to create in situ acute regional myocardial ischemia in a rabbit model for non-survival and survival experiments. The initial goal of this method was to evaluate the effects of mitochondrial transplantation on modulating myocardial necrosis and increasing the post-ischemic heart function following an ischemic event. Previous research has demonstrated the occurrence of mitochondrial alterations and a rapid decline in high-energy phosphate levels following the onset of ischemia and a reduction in the oxygen supply, resulting in a drastic decrease in the cardiac energy stores4. Investigators have attempted to improve post-ischemic function and lessen myocardial tissue necrosis using pharmacological interventions and/or procedural techniques, but these techniques provide limited cardioprotection and have minimal impact on mitochondrial damage and dysfunction5,6,7. Our team and others have previously shown that mitochondrial damage primarily occurs during ischemia and that contractile recovery can be enhanced and the myocardial infarct size decreased with the preservation of mitochondrial respiratory function during reperfusion8,9,10. Thus, we hypothesized that mitochondrial transplantation from tissues unaffected by ischemia to the area of ischemia prior to reperfusion would provide an alternative approach to reduce myocardial necrosis and enhance myocardial function. Herein, we detail the protocol used to test this theory and the representative results obtained from our initial study analysis.
Furthermore, several investigators have focused on other topics integral to defining the impact of myocardial ischemia-reperfusion injury and establishing appropriate therapeutic interventions. One such area of research is that of preconditioning. Myocardial ischemic preconditioning is a cardioprotective mechanism activated by brief ischemic stress that results in a reduction in the rate of cardiac cell necrosis during subsequent episodes of prolonged ischemia. These mechanisms can be activated by either hypoxia or coronary occlusion. Mandel et al. demonstrated that hypoxic-hyperoxic preconditioning helped to maintain the balance of nitric oxide metabolites, reduced endothelin-1 hyperproduction, and supported organ protection11. Moreover, the concept of remote ischemic preconditioning, a phenomenon whereby single-organ preconditioning provides systemic protection, has been explored. Ali et al. found that, in patients undergoing elective open abdominal aortic aneurysm repair, remote preconditioning, performed by intermittently cross-clamping the common iliac artery to serve as a stimulus, reduced the incidence of postoperative myocardial injury, myocardial infarction, and renal impairment12.
Rabbit models offer potential advantages over models with other species and have been used in multiple different scenarios for decades, including the induction of arrhythmias, global and regional ischemic models, and cardiac contraction research, amongst others13,14,15. Although the rabbit heart is smaller than that of a dog or pig, it is large enough to easily perform surgical procedures at a much lower cost13. The rabbit heart is often used as it closely parallels the human heart; indeed, it has a similar metabolic rate, expresses &#946;-myosin heavy chain, andlacks significant myocardial xanthine oxidase16. The technique herein described to induce regional myocardial ischemia is simple, repeatable, and cost-effective. This method allows for both non-survival and survival cases, as only regional ischemia is induced rather than global ischemia, and the materials needed are non-specialized. Two different surgical approaches (i.e., sternotomy and mini-thoracotomy) can be utilized, thus providing the operator and experimental protocols more freedom in terms of the study design. Additionally, the procedure does not require the use of a cardiopulmonary bypass. In this context, minimally invasive approaches to coronary artery bypass grafting have become valuable alternatives for patients in need of multi-vessel revascularizaiton17,18. This model could be used to study the differences between these approaches and provide an animal-based learning tool for surgical trainees. Additionally, performing cardiac catheterization utilizing this model may be useful for physiological research and/or surgical training.
Our model provides a methodology for applications in which inducing regional myocardial ischemia and subsequently measuring the infarct size, myocardial function, and cellular changes are of importance. With this protocol, we have been able to evaluate several markers of cellular function and adaptation to ischemia and the proposed therapeutic intervention (i.e., mitochondrial transplantation) by examining the internalization of organelles, oxygen consumption, high-energy phosphate synthesis, and the induction of cytokine mediators and proteomic pathways. These outcomes are important in preserving the myocardial energetics, cell viability, and cardiac function and allow for the objective evaluation of cardioprotective techniques following ischemia-reperfusion injury. This model could be used to study similar biologic pathways and alternatives in the field of post-ischemic myocardial pathology and recovery.
The goal of this protocol is to provide a highly reproducible methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. This model provides a methodology with high survival, low intraoperative mortality, and minimal morbidity19. Other models for acute regional myocardial ischemia have been described using radiolabeled materials, contrast agents, magnetic resonance imaging, or computer simulations20,21,22. Our protocol provides a reliable and simple methodology that is cost-effective, consistently reproducible, and has a low technical demand and, thus, can be performed by investigators without surgical expertise. This protocol accommodates either a survival project using a left mini-thoracotomy or a non-survival model using a midline sternotomy.

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			<td>#10 blade</td>
			<td>Bard Parker</td>
			<td>371210</td>
			<td></td>
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			<td>#11 blade</td>
			<td>Fisher Scientific</td>
			<td>B3L</td>
			<td></td>
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			<td>22 G PIV needle</td>
			<td>BD Insyte</td>
			<td>381423</td>
			<td></td>
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		<tr>
			<td>Acepromazine</td>
			<td>VETONE</td>
			<td>NDC 13985-587-50</td>
			<td>0.5 mg/kg IM and IV</td>
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			<td>Aline pressure bag</td>
			<td>Infu-Stat</td>
			<td>2139</td>
			<td></td>
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			<td>Angiocath</td>
			<td>Becton Dickinson</td>
			<td>382512</td>
			<td></td>
		</tr>
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			<td>Arterial Catheter</td>
			<td>Teleflex</td>
			<td>MC-004912</td>
			<td></td>
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			<td>Atropine</td>
			<td>Hikma Pharmaceuticals</td>
			<td>NDC 0641-6006-01</td>
			<td>&#160;0.01 mg/kg IM</td>
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			<td>Betadine and 70% isopropyl alcohol</td>
			<td>McKesson</td>
			<td>NDC 68599-2302-6</td>
			<td></td>
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		<tr>
			<td>Blood gas machine</td>
			<td>Siemens</td>
			<td>MRK0025</td>
			<td></td>
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			<td>Bovie</td>
			<td>Valleylab</td>
			<td>E6008</td>
			<td></td>
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			<td>Bulldog clamps</td>
			<td>World Precision Instruments</td>
			<td>14119</td>
			<td></td>
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			<td>Bupivacaine</td>
			<td>Auromedics</td>
			<td>NDC 55150-249-50</td>
			<td>&#160;3 mg/kg IM</td>
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			<td>Butorphanol</td>
			<td>Roxane</td>
			<td>NDC 2054-3090-36</td>
			<td>0.5 mg/kg IM</td>
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			<td>Clear acetate sheet</td>
			<td>Oxford Instruments</td>
			<td>ID 51-1625-0213</td>
			<td></td>
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			<td>Clipers</td>
			<td>Andis</td>
			<td>AGC2</td>
			<td></td>
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			<td>DeBakey forceps</td>
			<td>Integra</td>
			<td>P6280</td>
			<td></td>
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			<td>Echocardiography machine</td>
			<td>Philips</td>
			<td>IE33 F1</td>
			<td></td>
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			<td>Electrocardiography machine</td>
			<td>Meditech</td>
			<td>MD908B</td>
			<td></td>
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		<tr>
			<td>Endotracheal tube</td>
			<td>Medline</td>
			<td>#922774</td>
			<td></td>
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			<td>Fentanyl</td>
			<td>West-Ward</td>
			<td>NDC 0641-6030-01</td>
			<td>1&#8211;4 &#181;g/kg transdermal patch</td>
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			<td>Formaldehyde solution 10%</td>
			<td>Epredia</td>
			<td>94001</td>
			<td></td>
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			<td>Glass plates</td>
			<td>&#160;United Scientific</td>
			<td>B01MUHX6MR</td>
			<td></td>
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			<td>Heparin Sodium</td>
			<td>Sagent</td>
			<td>NDC 69-0058-02</td>
			<td>1000U in 1 mL 3 mg/kg</td>
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		<tr>
			<td>Hot water blanket</td>
			<td>3M</td>
			<td>55577</td>
			<td></td>
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			<td>Isoflurane</td>
			<td>Penn Veterinary Supply, INC</td>
			<td>NDC 50989-606-15</td>
			<td>1%&#8211;3%</td>
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			<td>Ketamine</td>
			<td>Dechra</td>
			<td>NDC 42023-138-10</td>
			<td>10 mg/kg IV</td>
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		<tr>
			<td>Lab Chart 7 Acquisition Software</td>
			<td>Adinstruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lactated Ringer&#39;s solution</td>
			<td>ICUmedical</td>
			<td>NDC 0990-7953-09</td>
			<td>10 mL/kg/h</td>
		</tr>
		<tr>
			<td>Laryngoscope</td>
			<td>Welch Allyn</td>
			<td>68044</td>
			<td></td>
		</tr>
		<tr>
			<td>Left ventricule lumen catheter&#160;3Fr</td>
			<td>McKesson</td>
			<td>385764-EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine (1%)</td>
			<td>Pfizer</td>
			<td>4276-01</td>
			<td>1&#8211;1.5 mL/kg IV</td>
		</tr>
		<tr>
			<td>LVDP transducer</td>
			<td>Edward</td>
			<td>PDP-ED</td>
			<td></td>
		</tr>
		<tr>
			<td>Marking pen</td>
			<td>Viscot</td>
			<td>1451SR-100 Unsterile</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo scissors</td>
			<td>Mayo</td>
			<td>S7-1098</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Entireoly Pets Pharmacy</td>
			<td>NDC 015914-005-01</td>
			<td>0.25 mg/kg IM</td>
		</tr>
		<tr>
			<td>Metzenbaum scissors</td>
			<td>Cole-Parmer</td>
			<td>UX-10821-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Monastra. Blue pigment 98%</td>
			<td>Chemsavers</td>
			<td>MBTR1100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Monocryl 5-0</td>
			<td>Ethicon</td>
			<td>Y463G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito clamp</td>
			<td>Shioda</td>
			<td>802N</td>
			<td></td>
		</tr>
		<tr>
			<td>PDS 3-0</td>
			<td>Ethicon</td>
			<td>42312201</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezoelectric sonomicrometry crystals</td>
			<td>Sonometrics</td>
			<td>Small 2mm round</td>
			<td></td>
		</tr>
		<tr>
			<td>Plegets</td>
			<td>DeRoyal</td>
			<td>32-363</td>
			<td></td>
		</tr>
		<tr>
			<td>Povuine Iodine Prep Solutions</td>
			<td>Medline</td>
			<td>MDS093940</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision vaporized system face mask</td>
			<td>Yuwell</td>
			<td>B07PNH69BF</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolene 3-0</td>
			<td>Ethicon</td>
			<td>8665G</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline 5-0</td>
			<td>Ethicon</td>
			<td>8661G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse oximetry probe</td>
			<td>Masimo</td>
			<td>9216-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Rib spreader</td>
			<td>Medline</td>
			<td>MDS5621025</td>
			<td></td>
		</tr>
		<tr>
			<td>S12 Pediatric Sector Probe</td>
			<td>Phillips</td>
			<td>21380A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonomicrometer</td>
			<td>Sonometrics</td>
			<td>BZ10123724</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Medline</td>
			<td>3.00802E+13</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile towels</td>
			<td>McKesson</td>
			<td>MON 277860EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sternal retractor</td>
			<td>Medline</td>
			<td>MDS5610321</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures for closure</td>
			<td>J&#38;J Dental</td>
			<td>8698G</td>
			<td></td>
		</tr>
		<tr>
			<td>Telemetriy monitor</td>
			<td>Meditech</td>
			<td>MD908B</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature probe</td>
			<td>Omega</td>
			<td>KHSS-116G-RSC-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Triphenyl tetrazolium chloride (1%)</td>
			<td>Millipore</td>
			<td>MFCD00011963</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>MedGroup</td>
			<td>MSLGA 11</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl 2-0</td>
			<td>Ethicon</td>
			<td>V635H</td>
			<td></td>
		</tr>
		<tr>
			<td>Vinyl tubing</td>
			<td>ABE</td>
			<td>DISW 3001</td>
			<td></td>
		</tr>
	</tbody>
</table>

model of ischemia and reperfusion injury in rabbits

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer&#39;s solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis.
With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located.
A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse.
For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively.
For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

Following the protocol (Figure 1), myocardial ischemia was confirmed immediately by the direct visualization of cyanosis of the epicardium.
Standard ECGs (three limb leads: I, II, and III, and three computed augmented leads: aVL, aVR, and aVF) were recorded continuously pre-ischemia, during ischemia, and at reperfusion (Figure 2). The ECGs demonstrate tachycardia, arrhythmias (i.e., ventricular fibrillation), conduction system defects...

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Model of Ischemia and Reperfusion Injury in Rabbits

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and ...

This protocol provides a simple, reliable, and reproducible methodology to induce in situ acute regional myocardial ischemia and reperfusion injury in a rabbit model for non-survival and survival experiments. This technique has minimal mortality and morbidity via two different surgical approaches. The methodology is cost-effective and has low technical demand and thus can be performed by investigators without surgical expertise.Exposing the LAD through a mini-thoracotomy can be challenging. We recommend making a fourth intercostal space anterior parasternal incision extending five to six centimeters laterally and practicing such exposure and rabbit necropsy prior. To perform a left mini-thoracotomy for survival studies, elevate the left side of the anesthetized rabbit approximately 30 degrees using a pillow.Palpate and outline the bony landmarks with a felt-tipped marking pen. Using a number 10 blade, incise the skin overlying the fifth rib, extending from adjacent to the lateral border of the sternum to five to six centimeters laterally. Using electrocautery, divide the pectoralis major muscle medially, followed by the serratus anterior muscle laterally.Then divide the intercostal muscles above the fifth rib to preserve the neurovascular bundle. Using sharp dissection, incise the parietal pleura with Metzenbaum scissors, carefully enter the pleural space through the fourth intercostal space, and extend the initial pleural incision in both directions parallel to the rib until a rib spreader or sternal retractor can be inserted. Place a rib spreader or sternal retractor within the rib space and widen it to visualize the heart and pericardial sac.Use DeBakey forceps to lift the pericardium. Open it with Metzenbaum scissors and proceed with LAD artery isolation. This will provide optimal visualization of the anterolateral surface of the heart with adequate exposure to the LAD and its branches.To isolate the LAD artery, encircle its third diagonal branch with a 3-0 polypropylene suture on a taper needle, then remove the needle. In survival cases, trim the three 3-0 polypropylene thread used to isolate the LAD and loosely tie the ends together. The three 3-0 polypropylene thread is used to identify the area at risk and the infarct zone upon harvesting the heart.Post-surgery, tie two 2-0 Polyglactin 910 figure-of-eight stitches around the ribs. Close the muscular and subcutaneous layers with a 3-0 polydioxanone suture in a running fashion. Then close the skin in a subcuticular fashion using a 5-0 monofilament suture.Finally, perform a needle thoracentesis to evacuate the pleural error. For non-survival studies, immediately proceed with midline sternotomy using curved Mayo scissors. Using a sternal retractor to visualize the heart and pericardial sac, open the pericardium as previously demonstrated.Visualize the 3-0 polypropylene thread loop placed during the previously performed thoracotomy portion of the procedure. To isolate the LAD artery, encircle its third diagonal branch with a 3-0 polypropylene suture on a taper needle. Then remove the needle and thread both ends of the polypropylene filament through a small vinyl tube to form a snare.Using DeBakey forceps, place a rectangular PTFE pledget between the two polypropylene filaments, sandwiching it between the isolated LAD artery and the vinyl tube. Tighten this snare by pressing down on the vinyl tube and pulling up on the polypropylene suture filaments to occlude the coronal artery. Clamp the tube using a mosquito clamp to maintain the desired tightness.Confirm myocardial ischemia by observing regional cyanosis of the epicardium. Remove the snare after 30 minutes of ischemia induction. At the end of the experiment, harvest the heart for biochemical and tissue analysis.The recorded ECGs at pre-ischemia, during ischemia, and reperfusion demonstrated tachycardia, arrhythmias, conduction system defects, the development of infarct-related Q waves, and ST segment deviation. The 2D echo readings showed that the fractional shortening decreased during ischemia and post-ischemia compared to pre-ischemia. After 30 minutes of induced regional myocardial ischemia, evidence of myocardial necrosis was observed.The most critical protocol steps are carefully encircling the LAD without damaging the artery or causing venous bleeding, appropriately placing the PTFE pledget to prevent vasospasm, and including the LAD to create a consistent area at risk. This methodology is useful in vascular injury, chronic myocardial ischemia, and acute myocardial stunning models to unveil cardiovascular molecular mechanics, pathophysiological mechanisms, and therapeutic targets, demonstrating usability and developing diagnostic and treatment algorithms.

model-of-ischemia-and-reperfusion-injury-in-rabbits

Research

JoVE Journal

Medicine

922 ビュー.  Harvard Medical School. このプロトコルは非存続および存続の実験のためのウサギ モデルでその場の激しい地方心筋虚血そして再灌流の傷害を引き起こすために簡単で、信頼でき、再現性がある方法論を提供する。この技術は、2つの異なる外科的アプローチにより、死亡率と罹患率が最小限に抑えられています。この方法論は費用対効果が高く、技術的要求が低いため、外科の専門知識がなく...