This publication was supported by the South Carolina Clinical &#38; Translational Research (SCTR) Institute, with an academic home at the Medical University of South Carolina, NIH/NCATS Grant Number UL1 TR000062. Further support was provided by VA merit award BX002327-01 to DRM. DJH was supported by NIH/NCATS Grant Number TL1 TR000061 and by NIH Grant Number T32 GM008716. SEA was supported by NIH Grant Number T32 HL07260.

All procedures listed here have been approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. The experiments described here are acute, non-survival experiments. As such, there is no use of eye ointment and a sterile operating suite is not required. Euthanasia is achieved by exsanguination during harvesting of the heart.
1. Experimental Preparation
<ol>
	<li>Set up either a constant pressure or constant flow Langendorff Perfusion Apparatus4.</li>
	<li>Prepare 4 L of modified Krebs Henseleit Buffer (in mM: 112 NaCl, 5 KCl, 1.2 MgSO4, 1 K2HPO4, 1.25 CaCl2, 25 NaHCO3, 11 D-glucose, 0.2&#160;octanoic acid, pH = 7.4).
	<ol>
		<li>Make 10x stock buffers.
		<ol>
			<li>Dissolve NaCl, KCl, MgSO4, and K2HPO4 in 3 L ultrapure water.</li>
			<li>Dissolve CaCl2 in 500 ml ultrapure water with 40 ml of 1 N HCl.</li>
			<li>Slowly add CaCl2 solution to the buffer made in 1.2.1.1. Add enough ultrapure water to total 4 L. Allow to stir for 1 hr before filtering through a 0.8 &#181;m filter. Store at 4 &#176;C.</li>
			<li>Dissolve NaHCO3 in 4 L ultrapure water. Allow to stir for 1 hr before filtering through at 0.8 &#181;m filter. Store at 4 &#176;C</li>
		</ol>
		</li>
		<li>Combine 400 ml 10x Krebs Henseleit buffer with 400 ml 10x NaHCO3 buffer. Add enough ultrapure water to total 4 L. Dissolve glucose and octanoic acid while stirring. Filter through 0.8 &#181;m filter.</li>
	</ol>
	</li>
	<li>Prepare system for heart.
	<ol>
		<li>Add 2 L of filtered buffer to a 4 L bottle placed 38 in&#160;above the stopcock attached to the aortic cannula. This will deliver a perfusion pressure of 70-80 mmHg to the heart, which is the recommended perfusion pressure4.</li>
		<li>Insert gas dispersion bubbler into the buffer in the bottle.</li>
		<li>Run the buffer through the system, being sure to eliminate any bubbles from inside the tubing.</li>
		<li>Turn on gas (95% O2/5% CO2) at least 30 min before heart is to be placed on the system. Use a gas flow probe and blood gas analyzer to verify oxygen saturation.</li>
	</ol>
	</li>
	<li>Prepare surgical supplies and balloon.
	<ol>
		<li>For harvesting of heart, prepare one pair of 6.75&#8221; surgical scissors, one pair of 4.5&#8221; scissors, two pairs of hemostats, two sets of caramalt forceps, two syringes with 27 G needles, 2 pieces of 0-guage silk suture (about 15 cm in length), one 1/16 inch barbed cannula, and one tracheal cannula.</li>
		<li>To assemble tracheal cannula, glue a short piece of 1/16 inch diameter plastic tubing to a Y-connector.</li>
		<li>To assemble balloon apparatus: attach a gavage needle to a piece of 1/16 inch tubing, then attach tubing to pressure transducer housing. A small syringe attached to the other side of the pressure transducer housing will allow inflation and deflation of the balloon.
		<ol>
			<li>Cut a square of plastic wrap and place tip of a gavage needle in the center. Wrap the plastic wrap around the gavage needle.</li>
			<li>Fill balloon with saline and tie off with two sutures. Inflate balloon to ensure there are no leaks. The volume of saline within the inflated balloon should be roughly 200 &#181;l, but can vary depending on the size of the rat.</li>
			<li>Alternatively, use a latex finger cot in place of the plastic wrap. Place gavage needle within finger cot, fill with saline and tie off.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Harvest the heart
<ol>
	<li>Restrain a Sprague Dawley rat using a decapicone or other manual restraint device. Weigh rat.</li>
	<li>Administer ketamine/xylazine mixture (0.85 mg/kg ketamine and 0.15 mg/kg xylazine) via intraperitoneal injection to anesthetize rat.&#160; This dose of anesthetic will provide sufficient anesthesia for 20-40 min, but the procedure should be completed as quickly as possible after anesthesia is confirmed.</li>
	<li>Confirm proper degree of anesthesia by testing toe pinch reflex.</li>
	<li>Tracheotomy
	<ol>
		<li>Remove pelt from mid-forepaw to base of jaw, using hemostats to elevate pelt and scissors to make incision. Keep the pelt elevated to separate it from the underlying muscle, allowing one to cut along the pelt and remove it.</li>
		<li>Using hemostats, lift glandular tissue and make a horizontal incision in fascia inferior to the glandular tissue. Use hemostats to spread incision open, taking care not to nick the jugular veins.</li>
		<li>Using hemostats, pinch muscle overlying trachea, and using scissors make a transverse incision in the muscle just below the tip of the hemostats. Using hemostats, spread tissue apart to reveal trachea.</li>
		<li>Carefully insert hemostats under trachea, clearing fascia while progressing by gently advancing and retracting hemostats.</li>
		<li>Once hemostats are successfully placed behind the trachea, pinch 0-0 silk suture with hemostats and hemisect the trachea with a small, sharp pair of scissors.</li>
		<li>Insert tracheal cannula into the hemisected trachea, pull suture behind the trachea, and tie the suture around the cannulated trachea, anchoring the suture to the Y-groin of the cannula with a surgeons knot.</li>
		<li>Using a 27 G needle, inject 1,000 U/kg heparin into the jugular vein and allow to circulate for 30 sec.</li>
	</ol>
	</li>
	<li>Thoracotomy
	<ol>
		<li>Remove pelt from mid-abdomen to mid-forepaw region by pinching with hemostats and cutting with scissors.</li>
		<li>Make a &#190; inch transverse incision into the abdominal muscle wall, just below the diaphragm.</li>
		<li>Make a vertical incision up the abdominal wall and chest wall, cutting along the sternum.</li>
		<li>Cut the diaphragm back bilaterally, then spread ribcage to reveal heart, clamp ribcage with hemostats to ensure it remains spread.</li>
		<li>Using hemostats, gently remove thymus, exposing the ascending aorta.</li>
		<li>Tease hemostats through aortic loop, advancing and retracting hemostats and gently spreading fascia to allow tip of hemostats through.</li>
		<li>Use hemostats to pull 0-0 silk suture behind ascending aorta, tie suture into a loose half-square knot.</li>
		<li>Turn stopcock to start flow of perfusion buffer, hemisect aorta and immediately insert cannula into aortic lumen. Rapidly cinch a half-square knot and complete the knot, tightening thoroughly.</li>
		<li>Use scissors to dissect heart away from the great vessels, remove heart from chest and attach to perfusion column.</li>
		<li>Trim away excess lung tissue and allow heart to equilibrate on column for about 15 min before insertion of balloon. Use a normal flow rate of buffer through the heart of 10-20 ml/min.</li>
	</ol>
	</li>
</ol>
3. Langendorff Perfusion and Ischemia Reperfusion Injury
<ol>
	<li>Placement of LV pressure balloon
	<ol>
		<li>Deflate balloon while rolling into a cone shape and turn stopcock to ensure balloon remains deflated.</li>
		<li>Excise left atrium with a small pair of scissors to expose access to mitral valve.</li>
		<li>Insert balloon through left atrium and mitral valve into left ventricle.</li>
		<li>Start pressure monitoring software (LabChart Pro).</li>
		<li>Inflate balloon by opening stopcock and gently increasing amount of saline in balloon until diastolic pressure reading from micromanometer exceeds zero.</li>
		<li>To check length-tension response, inflate balloon slowly to a diastolic pressure of 75 mmHg, deflate slowly, repeat once.</li>
		<li>Set diastolic pressure to 10 mmHg by modulating the amount of saline in the balloon. Turn stopcock to seal balloon at this level of inflation, and maintain closed pressure system between LV balloon and pressure transducer.</li>
		<li>Lower heart into heated chamber and cover chamber with plastic.</li>
	</ol>
	</li>
	<li>Administration of Ischemia Reperfusion Injury
	<ol>
		<li>Plug the bottom of the heated chamber and allow chamber to fill with effused buffer.</li>
		<li>When buffer has submerged heart, turn stopcock to stop the flow of buffer into the heart, inducing global ischemia. Ischemia time can vary depending on the goals of the experiment.</li>
		<li>After 30 min of ischemia, turn stopcock to restore flow of buffer into heart, initiating reperfusion.</li>
		<li>Allow reperfusion of heart for 1 hr before terminating the experiment. Use variable reperfusion times, depending on the goals of the experiment.</li>
	</ol>
	</li>
	<li>Termination of Experiment and Harvesting of Tissue
	<ol>
		<li>Using side port of stopcock, administer 10 ml of phosphate buffered saline at 4 &#176;C into the heart at a rate of 10 ml/min to arrest the heart.</li>
		<li>Remove heart from perfusion column, trim away remaining atrial tissue.</li>
		<li>Place heart into a stainless steel tissue slicing matrix, cover with Parafilm, and place at -80 &#176;C for 8 min, or until the heart has a marshmallow-like consistency.</li>
		<li>Remove block from freezer and insert razorblades into slicing matrix to slice heart into 2 mm slices. Tissue blocks can also be purchased with slice sizes other than 2 mm.</li>
		<li>Remove slices one at a time and drop into liquid nitrogen to flash freeze for biochemical studies. Retain the third ventricular slice from the apex for infarct staining.</li>
		<li>Remove tissue from liquid nitrogen and store at -80 &#176;C until use in biochemical experiments.</li>
	</ol>
	</li>
	<li>Infarct Staining
	<ol>
		<li>Incubate the third ventricular slice in 10 ml of 1% w/v triphenyltetrazolium chloride (TTC) at 37 &#176;C for 15 min. 
		Note: Incubation in TTC can be performed for 15 or 30 min with no change in effectiveness15.</li>
		<li>Transfer tissue slice from TTC to 10% buffered formalin and allow to incubate at room temperature overnight. For optimal results, hearts should be taken out of formalin and imaged within 24 hr4.</li>
		<li>Photograph stained slice and use software such as ImageJ to estimate infarct size according to manufacturer&#8217;s protocol. Take photographs as soon as possible after fixation, as the dye will fade over time.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The isolated perfused rat heart can be successfully used to study the effect of pharmacological intervention on cardiac preconditioning in ischemia reperfusion injury9. However, there are some essential steps to the procedure that must be standardized in order to ensure reproducible results. Maintaining a temperature of 37.4 &#176;C within the system is critical, as even mild hypothermia and hyperthermia can cause cardiac preconditioning10,11. The overall time that elapses from injection of anesthetic to the excision of the heart must be kept to a minimum, as prolonged exposure to ketamine may interfere with cardiac preconditioning12. Timely cannulation of the aorta in situ is essential to preventing the development of hypoxia within the heart or the exsanguination of the animal prior to heart excision. Overall, the time from first incision to removal of the heart should be no longer than 6-8 min. The left ventricular balloon must be checked for leaks before each experiment, and replaced if necessary. The system must be meticulously maintained, including flushing the entire apparatus with distilled water after each run and replacing worn-out tubing as necessary to prevent leakage or contamination of the interior of the tubing.
The isolated heart may be modified for any number of novel uses, including fluorescence imaging13, NMR Spectroscopy7, and optical mapping14 among many others. The system can also be modified to perfuse hearts from different animals, including mice. This modification is especially useful as it allows for experiments using transgenic mice. To modify the system for mouse hearts, smaller cannulae and a smaller perfusion column must be used, in addition to other modifications. Detailed descriptions of how to utilize the Langendorff method for mouse hearts have been published elsewhere16,17. Other modifications of this protocol include the administration of various drugs at different time points. When investigating the ability of a drug to cause pharmacological preconditioning or post-conditioning, it is essential to administer the drug at different time points in relation to the ischemia reperfusion injury. The drug may be administered to the animal before the heart is excised, or mixed into the buffer either before the heart is placed on the column or during the ischemic phase so that the drug is present upon reperfusion. Alternatively, a side-port can be utilized to administer a bolus of drug at any time point during the protocol. Additionally, the protocol may be modified to change the duration of ischemia, reperfusion, or both. This allows the analysis of functional and biochemical data at multiple time points and can be used to track the time course of the acute effect of a drug. If the reperfusion period is altered, it is important to note that at least 60 minutes of reperfusion are necessary for TTC staining to effectively delineate infarct area15.
The main limitation of the isolated heart model in terms of ischemia reperfusion injury is that it does not account for many of the systemic factors that are present in the setting of ischemia reperfusion in vivo. This elimination of systemic influence must be accounted for in the analysis of data generated using the Langendorff model, but does not preclude the ability of the model to answer novel questions about the response of myocytes, fibroblasts, and endothelial cells to ischemia and subsequent reperfusion. The isolated heart model allows for complete manipulation of many of the variables affecting ischemia reperfusion injury in addition to real time analysis of the functional effects on the heart over short time intervals. The data generated by using the isolated heart system is invaluable in understanding the pharmacological preconditioning or post-conditioning of the heart in response to various pharmaceutical interventions.

Elucidation of the events underlying the cardiac response to both ischemia and reperfusion are essential in improving the treatment of myocardial infarction1 and cardiac surgical procedures that require aortic cross-clamping2. While in vivo models of ischemia reperfusion injury allow very useful endpoint analysis, they are not as effective for studying the functional effects of ischemia reperfusion injury acutely in real time. Additionally, in vivo ischemia reperfusion models generally produce significant variability in infarct size, and direct delivery of drug to the heart at the time of reperfusion is challenging. The utilization of a Langendorff isolated heart system for studying ischemia reperfusion injury allows for real-time functional assessment of pharmacological treatments, uniform area of infarcted tissue, and instantaneous delivery of drug directly to the myocardium.
First described by Oscar Langendorff in 18953, the Langendorff isolated heart is a robust model for studying ischemia reperfusion injury, having been used in ischemia reperfusion research for the last 40 years4,5. Here, some modifications are made to optimize the isolated heart for functional analysis. In situ cannulation of the aorta while the heart is beating ensures that the heart does not experience ischemic preconditioning, which would alter the results of ischemia reperfusion trials6. To facilitate this, a tracheotomy is performed, allowing ventilation and ensuring physiological stability of the rat during surgery. The heart is then attached to a glass water-jacketed spiral column through which Krebs Henseleit buffer is delivered via retrograde perfusion directly into the aorta. A saline-filled balloon is inserted into the left ventricle and attached to a pressure transducer, which allows for real time measurement of pressures from within the ventricle and calculation of multiple functional parameters. At the conclusion of the experiment, the heart is flushed with cold saline to arrest contraction and flash frozen in liquid nitrogen to enable downstream analysis of DNA, RNA and protein levels. Thus modified, the Langendorff perfused heart serves as an effective system for direct monitoring of the physiological effect of pharmacological interventions at any time acutely during the ischemia reperfusion injury.

<table><tbody><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>S3014</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>P9541</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma Aldrich</td><td>203726</td><td></td></tr><tr><td>Potassium Phosphate Dibasic</td><td>Sigma Aldrich</td><td>RES20765-A7</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma Aldrich</td><td>C8106</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma Aldrich</td><td>G8270</td><td></td></tr><tr><td>Octanoic Acid</td><td>Sigma Aldrich</td><td>C2875</td><td></td></tr><tr><td>2,3,5-triphenyltetrazolium chloride</td><td>Sigma Aldrich</td><td>T8877</td><td></td></tr><tr><td>Medical Pressure Transducer</td><td>MEMSCAP</td><td>SP844</td><td></td></tr><tr><td>Masterflex Peristaltic Pump</td><td>Cole Parmer</td><td>EW-07521-40</td><td></td></tr><tr><td>Masterflex Easy Load Pump Head</td><td>Cole Parmer</td><td>EW-07518-10</td><td></td></tr><tr><td>Heated circulating water bath</td><td>Lauda</td><td>M3</td><td></td></tr><tr><td>Tubing Flow Module</td><td>Transonic</td><td>TS410</td><td></td></tr><tr><td>Modular Research Console</td><td>Transonic</td><td>T402</td><td></td></tr><tr><td>Inline flow sensor</td><td>Transonic</td><td>ME2PXN</td><td></td></tr><tr><td>PowerLab Data Acquisition Device</td><td>AD Instruments</td><td>PL3508</td><td></td></tr><tr><td>LabChart data acquisition software</td><td>AD Instruments</td><td>MLU60/8</td><td></td></tr></tbody></table>

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 left ventricular balloon apparatus allows for real-time monitoring of the pressure developed by the contracting left ventricle (Figure 1). As described previously7, this pressure trace can be used to calculate many of the parameters of ventricular function. These calculations can be made in the baseline phase as well as the reperfusion phase, averaged over multiple traces within each group, and compared in order to determine whether the pharmacological intervention resulted in cardiac preconditioning, as we have done previously9. One such parameter is the developed pressure, calculated as the difference between the systolic pressure and the end diastolic pressure. The developed pressure in normal perfused rat hearts can range from 70 to 130 mmHg (Figure 1A). After an ischemic insult, the developed pressure is reduced and the end diastolic pressure elevates (Figure 1B). When the rats are administered a known preconditioning agent such as the class I and IIb HDAC inhibitor SAHA (vorinostat)18 prior to excising the heart, the reduction in developed pressure and the elevation of end diastolic pressure associated with ischemia reperfusion injury are attenuated (Figure 1C). Other measures of left ventricular function, such as the rate of pressure generation (dP/dtmax), the rate of pressure relaxation (-dP/dtmax), and the rate pressure product (RPP) can be directly obtained or calculated from the software output (Figure 2). The ischemic phase can also be monitored in real-time, with the apparent cessation of pressure generation within minutes of the onset of ischemia. Ischemic contraction can also be monitored by measuring the time to onset of contraction and the time to peak contraction.
Triphenyltetrazolium chloride (TTC) is commonly used to differentiate between metabolically active and inactive tissue, and is used here for infarct staining. Once absorbed into the tissue TTC is reduced by metabolic enzymes, turning the active tissue red. Inactive tissue does not reduce TTC, and as such will stain white8. Langendorff perfused rat hearts that have not been subjected to ischemic injury do not show any white areas (not shown), while the hearts subjected to ischemia reperfusion injury show substantial areas stained white, indicating infarcted tissue (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/52908/52908fig1.jpg" /> 
Figure 1: Representative pressure traces from left ventricular balloon. Pressure recorded by the LV balloon upon ventricular contraction. Hearts not subjected to ischemia (A) experience a minor loss of contractile ability over time. Hearts subjected to ischemia (B) show immediate loss of pressure generation, followed by tonic contraction. Upon reperfusion, these hearts experience elevated EDP and decreased developed pressure. Hearts preconditioned with SAHA and subjected to ischemia (C) show attenuation of ischemia reperfusion injury. N = 1 per group.
<img alt="Figure 2" src="/files/ftp_upload/52908/52908fig2.jpg" /> 
Figure 2: Calculated parameters of ventricular function. Rate of pressure generation (A), rate of pressure relaxation (B), developed pressure (C), and rate pressure product (D) are some parameters of ventricular function that can be calculated from the pressure monitoring software.
<img alt="Figure 3" src="/files/ftp_upload/52908/52908fig3.jpg" /> 
Figure 3: TTC staining for infarct area. Example ventricular cross-section after ischemia reperfusion injury. Dotted line delineates left ventricle (LV) from right ventricle (RV). Focal infarction is indicated by white area, whereas viable tissue is indicated by red-staining.

Watch this Scientific Journal Video about Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts at JoVE.com

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

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 ...

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Universidad San Sebastian

Research

JoVE Journal

Medicine

22.8K Views.  Medical University of South Carolina. 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.

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

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Biology

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Enhancing CVD Research with Modified Langendorff Perfusion

Modified Langendorff Perfusion for Extended Perfusion Times of Rodent Cardiac Grafts

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and scientific advancement. As a result, innovation, improvement and/or repurposing of the available research toolset can provide improved testbeds for research advancement. Langendorff perfusion is an extremely valuable research technique for the field of CVD research that can be modified to accommodate a wide array of experimental needs. This tailoring can be achieved by personalizing a large number of perfusion parameters, including perfusion pressure, flow, perfusate, temperature, etc. This protocol demonstrates the versatility of Langendorff perfusion and the feasibility of achieving longer perfusion times (4 h) without graft function loss by utilizing lower perfusion pressures (30-35 mmHg). Achieving extended perfusion times without graft damage and/or function loss caused by the technique itself has the potential to eliminate confounding elements from experimental results. In effect, in scientific circumstances where longer perfusion times are relevant to the experimental needs (i.e., drug treatments, immunological response analysis, gene editing, graft preservation, etc.), lower perfusion pressures can be key for scientific success.

Author Spotlight: Advancing Cardiovascular Research &#8212; Tailored Langendorff Perfusion Techniques for Improved Experimental Outcomes

This article demonstrates the feasibility of achieving longer perfusion times (4 h) of murine cardiac grafts without function loss by employing lower (30-35 mmHg) than physiological (60-80 mmHg) perfusion pressures during Langendorff.

Despite important advancements in the diagnosis and treatment of cardiovascular diseases (CVDs), the field is in urgent need of increased research and ...

Bioengineering

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

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 ...

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.

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 ...

Modified MCAO/R Model with Preserved ECA for Enhanced Reperfusion and Stability

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

Author Spotlight: A Novel Approach to Cerebral Ischemia Modeling &#8211; Enhancing Reperfusion and Simplifying Procedure

This protocol describes the process of preparing for middle cerebral artery occlusion reperfusion via the common carotid artery.