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 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.
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.
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
2. Harvest the heart
3. Langendorff Perfusion and Ischemia Reperfusion Injury
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).
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.
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.
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.
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 °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.
The authors have nothing to disclose.
This publication was supported by the South Carolina Clinical & 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.
Sodium Chloride | Sigma Aldrich | S3014 | |
Potassium Chloride | Sigma Aldrich | P9541 | |
Magnesium Sulfate | Sigma Aldrich | 203726 | |
Potassium Phosphate Dibasic | Sigma Aldrich | RES20765-A7 | |
Calcium Chloride Dihydrate | Sigma Aldrich | C8106 | |
Sodium Bicarbonate | Sigma Aldrich | S5761 | |
D-Glucose | Sigma Aldrich | G8270 | |
Octanoic Acid | Sigma Aldrich | C2875 | |
2,3,5-triphenyltetrazolium chloride | Sigma Aldrich | T8877 | |
Medical Pressure Transducer | MEMSCAP | SP844 | |
Masterflex Peristaltic Pump | Cole Parmer | EW-07521-40 | |
Masterflex Easy Load Pump Head | Cole Parmer | EW-07518-10 | |
Heated circulating water bath | Lauda | M3 | |
Tubing Flow Module | Transonic | TS410 | |
Modular Research Console | Transonic | T402 | |
Inline flow sensor | Transonic | ME2PXN | |
PowerLab Data Acquisition Device | AD Instruments | PL3508 | |
LabChart data acquisition software | AD Instruments | MLU60/8 |