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 sex; sexually mature 15-20 weeks old; 3-4 kg body weight) for experimental studies.</li>
	<li>Anesthetic and analgesic agents:
	<ol>
		<li>Use atropine at a dose of 0.01 mg/kg intramuscular (IM)</li>
		<li>Use acepromazine at a dose of 0.5 mg/kg IM for initial sedation and 0.5 mg/kg intravenous (IV) for full anesthetization.</li>
		<li>Use butorphanol at a dose of 0.5 mg/kg IM.</li>
		<li>Use isoflurane via a precision vaporized system face mask at 3% for induction, followed by intubation at 1%-2%, oxygen (O2) at 100% at 2 L/min, and general anesthesia at 1% for maintenance.</li>
		<li>Use medetomidine at a dose of 0.25 mg/kg IM.</li>
		<li>Use ketamine at a dose of 10 mg/kg IV.</li>
		<li>Use a bupivacaine intercostal block at the thoracotomy site at a dose not exceeding 3 mg/kg IM.</li>
		<li>Use 1% lidocaine at a dose of 1-1.5 mL/kg IV.</li>
		<li>Use a 1-4 &#181;g/kg fentanyl transdermal patch for 72 h.</li>
	</ol>
	</li>
</ol>
2. Procedural steps (Figure 1)
<ol>
	<li>Sedate New Zealand White adult rabbits with a single combined IM injection of atropine, acepromazine, and butorphanol. Induce the animal with 3% isoflurane via a precision vaporized system face mask.</li>
	<li>Preparation prior to blinded endotracheal intubation (i.e., without visualization of the glottis)
	<ol>
		<li>Spray the larynx with 1% lidocaine to prevent laryngospasm.</li>
		<li>Pre-measure the endotracheal tube (ETT) length on the outside of the rabbit from the teeth to the predicted carina, and place the rabbit in a sternal recumbency position with the neck extended.</li>
	</ol>
	</li>
	<li>Intubate the animal with a cuffed pediatric size (3-0 or 3-5 inner diameter) ETT under continuous inhaled anesthetic at 1%-2% and O2 at 100% at 2 L/min.
	<ol>
		<li>Insert the ETT into the mouth, and direct it past the torus into the pharynx.</li>
		<li>Advance the ETT until either the tip of the tube contacts the glottis or the breath sounds are lost, indicating the tube tip has passed through the glottic opening.</li>
		<li>Slightly withdraw the tube until the breath sounds are regained, and then re-advance again, and secure the tube in place.</li>
	</ol>
	</li>
	<li>Ventilate the animal with mechanical support (tidal volume: 10 mL/kg, fraction of inspired O2: 40%, respiratory rate: 30-40 breaths/min, positive end-expiratory pressure: 5-10 cmH2O).
	<ol>
		<li>Adjust the FiO2 as tolerated to achieve an O2 saturation greater than 92% as measured by pulse oximetry to prevent hyperoxia, which can provoke a systemic inflammatory response.</li>
	</ol>
	</li>
	<li>Verify the proper placement of the ETT by a physical exam (i.e., auscultation), clinical signs (i.e., observation of condensation at the end of the endotracheal tube), and with objective measures (i.e., end-tidal carbon dioxide).</li>
	<li>After approximately 10 min, deliver an IM injection of medetomidine to the rabbit to provide simultaneous anesthetic and analgesic effects.</li>
	<li>Maintain the general anesthesia with 1% isoflurane for the duration of the surgical procedure.</li>
	<li>Insert a 22 G IV catheter into the marginal ear vein, and secure it with tape to obtain peripheral IV access. 
	NOTE: The femoral vein can be used as an alternative site of venous access.
	<ol>
		<li>Fully anesthetize the animal with acepromazine IV and ketamine IV.</li>
		<li>Prior to incision, inject 1,000 U/mL heparin at a dose of 3 mg/kg IV.
		<ol>
			<li>Administer 1,000 U/mL heparin at a dose of 3 mg/kg initially, and re-dose hourly until the end of the experiment to maintain an activated clotting time of &#62;400 s, in keeping with the current surgical protocol.</li>
		</ol>
		</li>
		<li>Administer 1% lidocaine IV and/or epicardial asynchronized defibrillation as needed if ventricular fibrillation occurs during the surgery. Ventricular fibrillation usually stops with one or two doses of lidocaine.</li>
		<li>Perfuse lactate Ringer&#39;s solution continuously at 10 mL/kg/h. 
		NOTE: Given the small volume of fluids administered and the short operative times, the animals in survival studies in this work did not require diuresis prior to extubation or during the recovery period. If the animal develops a worsening pulmonary status (i.e., increasing the ventilator settings, evidence of pulmonary edema on auscultation, etc.), diuresis is advised.</li>
	</ol>
	</li>
	<li>Perform a carotid cut down, and place a 4 or 5 French arterial line to facilitate the intra-operative monitoring of the arterial blood pressure (BP). 
	NOTE: The femoral artery can be used as an alternative site of arterial access.</li>
	<li>Monitor and record all the physiologic and mechanical variables by continuous real-time analysis.
	<ol>
		<li>Monitor the arterial BP with the carotid arterial line, and record the O2 saturation using pulse oximetry via a sensor placed on a shaved paw.</li>
		<li>Monitor with an electrocardiogram (ECG) with three limb leads: I, II, and III, and three computed augmented leads: aVL, aVR, and aVF.
		<ol>
			<li>Record the ECG tracings at the pre-ischemic baseline, during ischemia, during reperfusion, and serially during days 7-28 of recovery (if performing a survival study).</li>
		</ol>
		</li>
		<li>Monitor the level of sedation by continuous monitoring of the BP and heart rate (HR).</li>
		<li>Monitor the temperature with a rectal probe.</li>
		<li>Use 2D ECHO from the left parasternal and apical views to assess the myocardial function at desired time points in both the survival and non-survival cases.
		<ol>
			<li>Assess the myocardial function using the fractional shortening (FS) by measuring the left ventricle end-diastolic distance (LVEDD) and the left ventricle end-systolic distance (LVESD) and using the following formula: 
			FS = (LVEDD &#8722; LVESD)/LVEDD &#215; 100</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>During surgery, place the animal on a circulating hot water blanket to maintain a stable core body temperature.</li>
	<li>Prepare and drape the animal in a sterile fashion:
	<ol>
		<li>Shave the surgical site, and prep with betadine and 70% isopropyl alcohol, each applied in triplicate. Pat the area dry with sterile gauze pads, and drape the entire animal with sterile towels.</li>
	</ol>
	</li>
	<li>Left mini-thoracotomy (survival studies)
	<ol>
		<li>Perform an intercostal block at the predetermined thoracotomy site with bupivacaine IM.</li>
		<li>Administer 1% lidocaine IV via the auricular vein prior to incision.</li>
		<li>Perform a left mini-thoracotomy through the fourth intercostal space along the upper portion of the fifth rib to avoid the neuromuscular bundle, which is located parallel to the undersurface of each rib.
		<ol>
			<li>Perform an anterolateral thoracotomy for the best visualization of the anterolateral surface of the heart (i.e., the anatomic location of the LAD diagonal branches).</li>
			<li>Position the rabbit with the left side elevated approximately 30&#176; using a pillow or bean bag.</li>
			<li>Secure the rabbit&#39;s ipsilateral leg above its head to create room for both the operative field and between the rib spaces.</li>
			<li>Palpate and outline the bony landmarks, including the ribs, sternum, and scapula, with a felt-tipped marking pen. Incise the skin overlying the fifth rib using a #10 blade. Ensure that the incision remains parallel to the rib.</li>
			<li>Use electrocautery to divide the pectoralis major muscle and serratus anterior muscle. Divide the intercoastal muscles right above the fifth rib with electrocautery to preserve the neurovascular bundle.</li>
			<li>Cautiously enter the pleural space through the fourth intercostal space with sharp or blunt dissection. Extend the initial pleural incision parallel to the rib in both directions with sharp or blunt dissection until a rib spreader or sternal retractor can be inserted.</li>
		</ol>
		</li>
		<li>Place a rib spreader or sternal retractor within the rib space, and widen to provide adequate visualization of the heart and pericardial sac. Lift the pericardium with DeBakey forceps, and open the pericardium with Metzenbaum scissors.</li>
		<li>LAD artery isolation
		<ol>
			<li>Encircle the second or third diagonal branch of the LAD artery with a polypropylene suture (3-0) on a taper needle. Remove the needle, and thread both ends of the polypropylene filament through a small vinyl tube to form a snare.</li>
			<li>Place a pledget between the snare and the coronary artery to avoid damaging the coronary and/or causing vasospasm with ligation.
			<ol>
				<li>Using DeBakey forceps, pick up a rectangular PTFE felt pledget (approximately 7 mm x 3 mm). Place the pledget between the two polypropylene filaments so that it is sandwiched between the isolated LAD artery and the vinyl tube when the snare is tightened.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Midline sternotomy (non-survival studies) 
	NOTE: The midline sternotomy approach is ideal for non-survival cases, for which more invasive monitoring with LVDP and sonomicrometry can be utilized.
	<ol>
		<li>Perform a midline sternotomy using curved Mayo scissors. Place a sternal retractor, and widen it to provide adequate visualization of the heart and pericardial sac.</li>
		<li>Lift the pericardium with DeBakey forceps, and open the pericardium with Metzenbaum scissors.</li>
		<li>Placing the three piezoelectric sonomicrometry crystals:
		<ol>
			<li>Make three small 1 mm cuts on the epicardium of the LV, forming the corners of a triangle. Place the piezoelectric sonomicrometry crystals inside the epicardium cuts.</li>
			<li>Secure the wires to the cardiac surface with a 5-0 polypropylene U-stitch. When recording using sonomicrometry, pause the mechanical ventilation to allow for an accurate recording over two to three heartbeats. 
			NOTE: If the heart fibrillates, 1% lidocaine is not effective, and epicardial defibrillation is needed, turn off the sonomicrometer, and disconnect it from the data acquisition system to protect both from electrical input.</li>
		</ol>
		</li>
		<li>LAD artery isolation:
		<ol>
			<li>Encircle the second or third diagonal branch of the LAD artery with a polypropylene suture (3-0) on a taper needle.</li>
			<li>Remove the needle, and thread both ends of the polypropylene filament through a small vinyl tube to form a snare.</li>
			<li>Place a pledget between the snare and the coronary artery to avoid damaging the coronary artery and/or causing vasospasm with ligation.</li>
			<li>Using DeBakey forceps, pick up a rectangular PTFE felt pledget (approximately 7 mm x 3 mm). Place the pledget between the two polypropylene filaments so that it is sandwiched between the isolated LAD artery and the vinyl tube when the snare is tightened.</li>
		</ol>
		</li>
		<li>Measurement of the LVDP:
		<ol>
			<li>Place a 5-0 polypropylene U-stitch in the apex of the LV. Make a small 1 mm incision with an 11 blade into the LV apex.</li>
			<li>Insert a 3 French balloon catheter into the LV lumen. Secure the catheter to the LV by tying it to the 5-0 polypropylene U-stitch suture.</li>
			<li>Connect the catheter to the transducer connected to the monitor to record the LVDP. Record the LVDP using the data acquisition system (described below). Zero the catheter to record the hemodynamic variables by opening the three-way stopcock to the air and zeroing on the monitor.</li>
		</ol>
		</li>
		<li>Data acquisition system
		<ol>
			<li>Initiate the data acquisition system (see the Table of Materials) on the computer/laptop being used. Connect the wire from the monitor to the computer/laptop.</li>
			<li>Select Channel 1 on the data acquisition system, and name it LVDP. Zero the transducer using the monitor. 
			NOTE: If connecting the BP and HR to the data acquisition system, follow the same process: connect the wire to the laptop, select Channel, and zero if measuring the BP.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Occlude the coronary artery by tightening the snare by pressing down on the vinyl tube while pulling up on the polypropylene suture filaments. Maintain the desired tightness with a mosquito clamp by directly clamping the tube and fixing it in place.</li>
	<li>Confirm myocardial ischemia visually by the regional cyanosis of the epicardium. Regional ischemia can also be confirmed on ECG with the presence of an ST segment and T wave changes.</li>
	<li>After visual confirmation, induce regional ischemia for 30 min under anesthesia.
	<ol>
		<li>At 0 min, 10 min, 20 min, and 30 min during the regional ischemia, assess the FS by 2D ECHO for both the survival and non-survival cases.</li>
		<li>Assess the LVDP and sonomicrometry continuously during the pre-ischemia time, myocardial ischemia time, and post-ischemic time for the non-survival cases.</li>
		<li>If needed, delineate the area at risk by ligating the artery again with the polypropylene suture stitch left in place. Cross-clamp the aorta, and inject Monastral Blue pigment 98% (diluted 1:5 in PBS) through the aorta using a cardioplegia needle. The perfused areas of the myocardium will stain blue, and the area at risk will remain unstained.</li>
		<li>Continuously monitor and record the HR, BP, and O2 saturation.</li>
		<li>Allow the animal to recover for 2 h (non-survival) or 28 days (survival). 
		NOTE: ECG can be used to confirm reperfusion. Although not seen in the experiment conducted in this study, hypokalemia can often occur during reperfusion and can be corrected with potassium control or an appropriate infusion.</li>
	</ol>
	</li>
	<li>Conclusion of the procedure
	<ol>
		<li>Survival cases
		<ol>
			<li>In survival cases, trim the 3-0 polypropylene thread used for the snare, loosely tie the ends together, and leave it in place. Identify the area at risk and the infarct zone by the 3-0 polypropylene thread.</li>
			<li>After the procedure is completed, close the incision in three layers.
			<ol>
				<li>Close the first layer by tying two 2-0 polyglactin 910 figure-of-eight stitches around the ribs.</li>
				<li>Close the muscular and subcutaneous layers with a 3-0 polydioxanone suture in a running fashion.</li>
				<li>Close the skin in a subcuticular fashion using a 5-0 monofilament suture. Use a buried running suture to minimize the irritation felt by the animal.</li>
			</ol>
			</li>
			<li>Evacuate the pleural air by performing a needle thoracentesis.</li>
			<li>Apply a fentanyl transdermal patch for 72 h to facilitate the postoperative pain management.</li>
			<li>Perform transthoracic echocardiography at the 1 week and 2 week time points postoperatively to assess the trends in the FS.</li>
			<li>Following the predetermined recovery period, sedate, intubate, and anesthetize the animal as above. Perform a median sternotomy. Expose and open the pericardial sac. Euthanize the rabbit under deep anesthesia by removing the heart en bloc,&#160;allowing the animal to expire by exsanguination.</li>
		</ol>
		</li>
		<li>Non-survival cases
		<ol>
			<li>After the experiment and having assured deep anesthesia, completely expose the heart, and remove it en bloc for biochemical and tissue analysis. The animal expires by exsanguination.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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<li>Sinusas, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+area+at+risk+during+coronary+occlusion+and+degree+of+myocardial+salvage+after+reperfusion+with+technetium-99m+methoxyisobutyl+isonitrile%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">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/pubmed?term=((Transplantation+of+autologously+derived+mitochondria+protects+the+heart+from+ischemia-reperfusion+injury%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury.</a> American Journal of Physiology. Heart and Circulatory Physiology. 304 (7), H966-H982 (2013).</li>
<li>Pombo, J. F., Troy, B. L., Russell, R. O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Left+ventricular+volumes+and+ejection+fraction+by+echocardiography%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Left ventricular volumes and ejection fraction by echocardiography.</a> Circulation. 43 (4), 480-490 (1971).</li>
<li>McCully, J. D., Wakiyama, H., Hsieh, Y. J., Jones, M., Levitsky, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+contribution+of+necrosis+and+apoptosis+in+myocardial+ischemia-reperfusion+injury%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Differential contribution of necrosis and apoptosis in myocardial ischemia-reperfusion injury.</a> American Journal of Physiology. Heart and Circulatory Physiology. 286 (5), H1923-H1935 (2004).</li>
<li>Masuzawa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+autologously+derived+mitochondria+protects+the+heart+from+ischemia-reperfusion+injury%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury.</a> American Journal of Physiology. Heart and Circulatory Physiology. 304 (7), 966-982 (2013).</li>
<li>Abarbanell, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+myocardial+and+vascular+injury%5BTitle%5D)+AND+%22Journal+of+Surgical+Research%22%5BJournal%5D)">Animal models of myocardial and vascular injury.</a> Journal of Surgical Research. 162 (2), 239-249 (2010).</li>
<li>Pirat, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+feature-tracking+echocardiographic+method+for+the+quantitation+of+regional+myocardial+function%3A+Validation+in+an+animal+model+of+ischemia-reperfusion%5BTitle%5D)+AND+%22Journal+of+the+American+College+of+Cardiology%22%5BJournal%5D)">A novel feature-tracking echocardiographic method for the quantitation of regional myocardial function: Validation in an animal model of ischemia-reperfusion.</a> Journal of the American College of Cardiology. 51 (6), 651-659 (2008).</li>
<li>Verdouw, P. D., vanden Doel, M. A., de Zeeuw, S., Duncker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+in+the+study+of+myocardial+ischaemia+and+ischaemic+syndromes%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">Animal models in the study of myocardial ischaemia and ischaemic syndromes.</a> Cardiovascular Research. 39 (1), 121-135 (1998).</li>
<li>Bolukoglu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+animal+model+of+chronic+coronary+stenosis+resulting+in+hibernating+myocardium%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">An animal model of chronic coronary stenosis resulting in hibernating myocardium.</a> The American Journal of Physiology. 263, H20-H29 (1992).</li>
<li>Heyndrickx, G. R., Millard, R. W., McRitchie, R. J., Maroko, P. R., Vatner, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regional+myocardial+functional+and+electrophysiological+alterations+after+brief+coronary+artery+occlusion+in+conscious+dogs%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Regional myocardial functional and electrophysiological alterations after brief coronary artery occlusion in conscious dogs.</a> Journal of Clinical Investigation. 56 (4), 978-985 (1975).</li>
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No conflicts of interest, financial or otherwise, are declared by the authors.

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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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&amp;amp;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...

Watch this Scientific Journal Video about Model of Ischemia and Reperfusion Injury in Rabbits at JoVE.com

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 ...

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

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1.1K Views.  Harvard Medical School. 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. 

Video: Model of Ischemia and Reperfusion Injury in Rabbits

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains ...

Developing an Experimental Aneurysm Model in a Rabbit Using an Elastase-Treated Vessel Pouch

<a href='https://app.jove.com/v/61157/arterial-pouch-microsurgical-bifurcation-aneurysm-model-in-the-rabbit'>Source: Wanderer, S., et al. Arterial Pouch Microsurgical Bifurcation Aneurysm Model in the Rabbit. J. Vis. Exp. (2020)</a>This video demonstrates the creation of an experimental aneurysm model in a rabbit using an elastase-treated vessel pouch. After excising a segment of the right CCA, the pouch is treated with elastase, then sutured to the left CCA to restore blood flow. The resulting stress on the weakened pouch walls induces aneurysm formation.