The present protocol describes aortic cannulation and retrograde perfusion of the ex-vivo neonatal murine heart. A two-person strategy, using a dissecting microscope and a blunted small gauge needle, permits reliable cannulation. Quantification of longitudinal contractile tension is achieved using a force transducer connected to the apex of the left ventricle.
The use of the ex-vivo retrograde perfused heart has long been a cornerstone of ischemia-reperfusion investigation since its development by Oskar Langendorff over a century ago. Although this technique has been applied to mice over the last 25 years, its use in this species has been limited to adult animals. Development of a successful method to consistently cannulate the neonatal murine aorta would allow for the systematic study of the isolated retrograde perfused heart during a critical period of cardiac development in a genetically modifiable and low-cost species. Modification of the Langendorff preparation enables cannulation and establishment of reperfusion in the neonatal murine heart while minimizing ischemic time. Optimization requires a two-person technique to permit successful cannulation of the newborn mouse aorta using a dissecting microscope and a modified commercially available needle. The use of this approach will reliably establish retrograde perfusion within 3 min. Because the fragility of the neonatal mouse heart and ventricular cavity size prevents direct measurement of intraventricular pressure generated using a balloon, use of a force transducer connected by a suture to the apex of the left ventricle to quantify longitudinal contractile tension is necessary. This method allows investigators to successfully establish an isolated constant-flow retrograde-perfused newborn murine heart preparation, permitting the study of developmental cardiac biology in an ex-vivo manner. Importantly, this model will be a powerful tool to investigate the physiological and pharmacological responses to ischemia-reperfusion in the neonatal heart.
Ex-vivo heart preparations have been a staple of physiologic, pathophysiologic, and pharmacologic studies for over a century. Stemming from the work of Elias Cyon in the 1860s, Oskar Langendorff adapted the isolated frog model for retrograde perfusion, pressurizing the aortic root to provide coronary flow with an oxygenated perfusate1. Using his adaptation, Langendorff was able to demonstrate a correlation between coronary circulation and mechanical function2. The ex-vivo retrograde perfused heart, later eponymously dubbed the Langendorff technique, has remained a cornerstone of physiologic investigation, leveraging its simplicity to powerfully study the isolated heart in the absence of potential confounders. The Langendorff preparation has been modified further to permit the heart to eject (the so-called "working heart") and allow the perfusate to recirculate3. However, the primary physiologic endpoints of interest have remained unchanged. Such endpoints include measures of contractile function, electrical conduction, cardiac metabolism, and coronary resistance4.
To evaluate cardiac function in his original frog heart preparation, Langendorff measured the tension generated by ventricular contraction in the longitudinal axis using a suture connected between the heart's apex and a force transducer.5 Isometric contraction was quantified in this manner with basal tension applied to the heart in the absence of ventricular filling. Refinement of the approach has led to fluid-filled balloons placed into the left ventricle via the left atrium to evaluate myocardial performance during isovolumic contraction6. To assess cardiac rhythm and the heart rate, surface leads can be placed on the poles of the heart to enable investigators to record the electrocardiogram. However, relative bradycardia can be expected, given the obligatory denervation. Extrinsic pacing may serve to overcome this and eliminate heart rate variability between experiments1. Another outcome measure, myocardial metabolism, can be assessed by measuring the oxygen and metabolic substrate content in the coronary perfusate and effluent and calculating the difference between them7. Lactate quantification in the coronary effluent can aid in characterizing periods of anaerobic metabolism as is seen with hypoxia, hypoperfusion, ischemia-reperfusion, or metabolic perturbations7.
Langendorff's original work enabled the study of the ex-vivo mammalian heart, using cats as the primary subject5. Evaluation of the isolated rat heart gained popularity in the mid-1900s with Howard Morgan, who detailed the 'working heart' rat model in 19675. The use of mice began only 25 years ago due to the technical complexity, tissue fragility, and relatively small murine heart size. Despite the challenges associated with mice study, the lower costs and ease of genetic manipulation have increased the appeal and demand of such murine ex-vivo preparations. Unfortunately, the application of the technique has been limited to adult animals, with juvenile 4-week-old mice being the youngest subjects utilized for ex-vivo study until quite recently8,9. While juvenile mice are "relatively immature" compared with adults, their utility as subjects for developmental biology studies is limited because they have, by and large, weaned from their birth dam and will soon begin puberty10. Adolescence occurs well beyond the postnatal transition in myocardial substrate utilization from glucose and lactate to fatty acids11. Thus, most information about the metabolic changes in the neonatal heart has historically resulted from ex-vivo work in larger species such as rabbits and guinea pig11.
Indeed, alternative approaches to the Langendorff preparation exist. These include in vitro experimentation, which lacks the whole organ functional data and context, or in vivo studies. This can be technically challenging and complicated by confounding variables such as the cardiovascular and respiratory effects of a requisite anesthetic agent, the influence of neurohumoral input, the consequences of core temperature, the nutritional status of the animal, and substrate availability12,13. Because the Langendorff approach permits the study of the isolated-perfused heart in an ex-vivo manner in a more controlled manner in the absence of such confounders, it has been and continues to be considered a powerful investigational tool. Therefore, the technique presented here gives researchers an experimental approach for the ex-vivo study of the newborn murine heart and limits time to reperfusion.
Investigating the heart during periods of development is an important consideration given the wide-ranging biochemical, physiologic, and anatomical transitions that occur during myocardial maturation. Shifts from anaerobic metabolism to oxidative phosphorylation, changes in substrate utilization, and progression from cell proliferation to hypertrophy are dynamic processes that uniquely occur in the immature heart11,14. Another critical aspect of the developing heart is that stressors encountered during necessary periods may produce heightened responses in the newborn heart and alter future susceptibility to insults in adulthood15. Although prior work has utilized newborn rats, lambs, and rabbits to study the Langendorff-perfused neonatal heart, advances permitting mice use are necessary given the importance of this species to developmental biology research16. To address this need, the first murine Langendorff-perfused newborn heart model using 10-day old animals was recently established6. Presented here is a method to enable successful aortic cannulation and establish retrograde perfusion of the isolated newborn murine heart. This approach may be utilized for pharmacology, ischemia-reperfusion, or metabolism studies focusing on whole organ function or can be adapted for the isolation of cardiomyocytes.
Institutional Animal Care and Use Committee of Columbia University Medical Center's approvals were obtained for all methods described. Wild-type C57Bl/6 male postnatal day 10 mice were used for the study.
1. Preparation of Langendorff apparatus
2. Fabrication of aortic cannula
3. Organ harvesting
4. Cannulation
5. Functional measurement
P10 mice were used to model a timepoint in human infancy26,27. Fifteen isolated C57Bl/6 newborn mouse hearts were harvested and cannulated successfully. Hearts were perfused with a continuous flow of 2.5 mL min-1 of warmed oxygenated KHB. Metabolic parameters, including glucose extraction, oxygen consumption, lactate production, and physiological parameters such as heart rate, perfusion pressure, and coronary resistance, were measured. Surface electrodes were used to record a continuous electrocardiogram, which allowed determining intrinsic rate and rhythm (Figure 2). Contractile force in the longitudinal axis was determined using the method described by Langendorff 28.
The metabolic assessment was performed to assess for adequacy of perfusion. Percent oxygen extraction was calculated by subtracting oxygen content in coronary effluent from the perfusate. Myocardial oxygen consumption was determined by multiplying coronary flow rate by the difference in oxygen content between the perfusate and coronary effluent multiplied by the solubility of oxygen (assuming 24 µL/mL of H2O at 37 °C and 760 mmHg)29,30. Using these calculations, it was determined that this perfusion strategy met the metabolic needs of the newborn mouse heart, given the negligible lactate production and low percent oxygen extraction and glucose consumption (Table 1).
All hearts beat spontaneously in sinus rhythm (Figure 2). As expected, however, the mean denervated intrinsic heart rate was slower than newborn murine heart rates reported in vivo31. Mean observed aortic perfusion pressures correlated well with the mean arterial pressures described in neonatal mice32. Other physiologic variable means were recorded and calculated (Table 2).
Based upon the observational data, exclusionary criteria to ensure consistency of the neonatal preparation needs to be considered (Table 3). A factor that is critical to the robustness of the preparation is the time required to initiate reperfusion. Cannulation is by far and away the most challenging step of the procedure, given the minuscule size and fragility of the neonatal mouse aorta. A prolonged delay in establishing cannulation or initiating reperfusion will injure the healthy heart or even precondition the myocardium1. Thus, minimizing the ischemic time to under 4 min is suggested (consistent with guidelines for the adult rodent heart)1. Following successful cannulation, assessment of the adequacy of perfusion is paramount. Signs of inadequate myocardial perfusion include prolonged arrhythmias, heart rate extremes, or aortic perfusion pressure extremes.
Figure 1: Aortic cannulation setup. (A) The proximal end of high-pressure tubing is attached to the "usual" cannula position site (shown in B). The cannula is attached to the distal end of the tubing (magnified in C). (B) "Usual" cannula position in apparatus. (C) The cannula is attached to the "slip-tip" end of the high-pressure tubing for ease of removal. Please click here to view a larger version of this figure.
Figure 2: The ex-vivo retrograde-perfused neonatal mouse heart. Image of a 10-day postnatal mouse heart after successful aortic cannulation with a 26 G blunt needle. Coronary effluent can be seen dripping from the heart through an incision in the right atrium. Stainless steel surface electrodes were placed at the poles to measure electrocardiogram continuously. Representative ECG tracing is displayed on the right in green, demonstrating a sinus rhythm and rate of 194 beats min-1. Not pictured is the suture connected between the apex of the heart and force transducer, allowing measurement of ventricular contractile force (waveform depicted in red on the right). Adapted with permission from Reference6. Please click here to view a larger version of this figure.
Metabolic parameter | Affluent | Effluent | Consumption | Extraction |
Glucose, mg·dL−1 | 194.3 ± 3.8 | 193.0 ± 5.5 | 1.4 ± 0.8 mg·dL−1 | |
Lactate, mmol·L−1 | < 0.3 ± 0.0 | < 0.3 ± 0.0 | ||
P02, mmHg | 641 ± 7.9 | 295 ± 18.4 | 28.2 ± 1.3 μL·min−1 | 55.7 ± 2.3% |
Table 1: Metabolic parameters of isolated perfused newborn murine hearts. Values are means ± SE. Affluent and coronary effluent was sampled, and PO2 (partial pressure of oxygen), glucose, and lactate were measured. Glucose uptake, oxygen extraction, and consumption were calculated. The difference in affluent and effluent determines extraction. Consumption is calculated as coronary flow x (PaO2– PvO2) x O2 solubility at 760 mmHg (assuming 24 µL/mL of H2O at 37 °C and 760 mmHg).
Physiologic Parameter | Mean |
Aortic perfusion pressure, mmHg | 47.9 ± 6.9 |
Coronary resistance, mmHg·min·mL−1 | 19.2 ± 2.8 |
Heart rate, beats·min−1 | 226 ± 8.9 |
Ventricular contractile force, g | 7.2 ± 1.2 |
Table 2: Physiologic parameters of isolated perfused newborn murine hearts. Values are means ± SE. Coronary resistance was calculated based on the coronary flow rate of 2.5 mL min-1 and aortic pressure using Ohm's law. According to Kirchoff's law of resistance in series, aortic pressure was calculated as pressure above baseline resistance in the system. Heart rate was measured via the surface electrode, and contractile force was measured via a suture connecting the apex of the heart to a force transducer. This table has been reprinted with permission from Reference6.
Physiologic Parameter | Exclusionary Threshold |
Time to reperfusion, min | >4 |
Aortic perfusion pressure, mmHg | <20 or >75 |
Heart rate, beats·min−1 | <150 or >300 |
Arrhythmia duration, min | >3 |
Table 3: Proposed exclusion criteria for neonatal murine heart Langendorff preparations. This table has been reprinted with permission from Reference6.
The present work describes successful aortic cannulation and retrograde perfusion in the isolated newborn mouse heart. Importantly, it allows researchers to overcome the barriers that young murine age and small heart size previously presented8. While not complex in design, the approach does require a significant degree of technical skill. Key steps that will inevitably challenge even the most technically proficient investigators will be cannulation of the aorta and securing the cannula in place. Difficulty with neonatal cannulation is not due solely to the small size of the aortic lumen. The relatively short length of the ascending aorta (aortic tissue between the aortic valve and right subclavian takeoff) may challenge investigators to precisely control the aortic cannula and necessitate careful coordination between teammates. Failure to appropriately position and secure the cannula within this region can ruin the preparation. For example, advancing the cannula too deep can damage the aortic valve or result in intraventricular cannulation. Placing the cannula too shallow within the aortic arch can lead to perfusate leakage out of one of the branches, such as the subclavian artery. Furthermore, forceful cannulation can tear the aorta. Such consequences of inartful cannulation will manifest with high flow rates or low perfusion pressures1. Alternatively, low flow rates or high perfusion pressures can indicate the presence of thrombi, air emboli, cannula occlusion, or coronary obstruction1. Arrhythmias, bradycardia, or tachycardia are all signs of inadequate perfusion regardless of etiology1,33.
A common and straightforward perfusion strategy should initially be chosen; constant flow using buffered crystalloid perfusate with glucose as a substrate in a spontaneously beating heart1. Adaptations to this approach will need to be assessed in future work and should include an assessment of the effect of different perfusion approaches and alternative perfusate and substrate strategies. While myocardial perfusion in this preparation was shown to be adequate for P10 hearts, the chosen flow rate might exceed the needs of the newborn heart. This is because the cardiac output in 10-day old mice is approximately 5.3 mL min-1 31. Thus, future work should investigate the effect of different flow rates and assess constant pressure strategies.
Constant pressure approaches may involve real-time flow adjustment mechanisms or a pop-off valve to limit maximal pressure5. This may be particularly important when studying ischemia-reperfusion injury, given the importance of evaluating coronary autoregulation in this context5. In addition, while intrinsic heart rate can be used as a biomarker for the adequacy of perfusion, pacing strategies are likely to be feasible and should be investigated in the future. Finally, future work should also assess alternative energy substrates in the oxygenated perfusate. This is because the newborn heart transitions from using glucose and lactate to consuming fatty acids in the neonatal period11,14. Thus, alternative metabolic substrates may be more physiologically relevant in this critical period of development.
Methodologic advances for evaluating murine cardiac function continue to emerge. Although the total number of research studies using the Langendorff preparation has remained consistent each year since the 1990s, the percentage of work utilizing murine-specific ex-vivo practices has steadily risen5. Thus, the importance of the isolated murine heart as a scientific model has increased over time. Innovations, such as the method described here, now permit the field to broaden the approach to the newborn mouse heart. In addition to its utility in ischemia-reperfusion research, such a method could also serve as an adjunct to other types of research techniques. For example, successful cannulation of the newborn mouse heart could facilitate cardiomyocyte isolation. To date, only 'chunk' digestion methods with lower yields have been available for isolating newborn mouse cardiomyocytes34. Therefore, the use of the neonatal Langendorff preparation with a retrograde infusion of enzymatic agents can improve the yield and quality of isolated cardiomyocytes35.
The neonatal response to ischemic injury is not equal to that of the adult, and the immature heart undergoes several transitions during the newborn period15,36. However, a better understanding of the developmental biology of the neonatal heart in health and disease is necessary. The differential effects of hypoxia, ischemia and reperfusion between neonatal and adult hearts have been investigated since the 1970s. However, these prior works have been limited to the use of animal species larger than the mouse37. The ability to generate transgenic mutants to study specific pathways and proteins of interest necessitates establishing a newborn murine ex-vivo preparation. The method detailed here enables successful aortic cannulation to establish retrograde perfusion of the isolated newborn murine heart. Using this approach, investigators will be able to study ischemia-reperfusion as it relates to the neonatal mouse. Such research will help us better understand the neonatal-specific protective mechanisms during ischemia, the newborn response to hypoxia, and the anatomic and metabolic developmental changes in the immature heart during health and disease states36,38,39. Therefore, the isolated perfused newborn heart model will prove to be a powerful tool for developmental cardiac biology research.
The authors have nothing to disclose.
NIH/NINDS R01NS112706 (R.L.)
Rodent Langendorff Apparatus | Radnoti | 130102EZ | |
24 G catheter | BD | 381511 | |
26 G needle on 1 mL syringe combo | BD | 309597 | |
26 G steel needle | BD | 305111 | |
5-0 Silk Suture | Ethicon | S1173 | |
Bio Amp | ADInstruments | FE135 | |
Bio Cable | ADInstruments | MLA1515 | |
CaCl2 | Sigma-Aldrich | C4901-100G | |
Circulating heating water Bath | Haake | DC10 | |
curved iris scissor | Medline | MDS10033Z | |
dissecting microscope | Nikon | SMZ-2B | |
find spring scissors | Kent | INS600127 | |
Force Transducer | ADInstruments | MLT1030/D | |
glucose | Sigma-Aldrich | G8270-100G | |
Heparin | Sagent | 400-01 | |
High pressure tubing | Edwards Lifesciences | 50P184 | |
iris dressing forceps | Kent | INS650915-4 | |
Jeweler-style curved fine forceps | Miltex | 17-307-MLTX | |
KCl | Sigma-Aldrich | P3911-25G | |
KH2PO4 | Sigma-Aldrich | P0662-25G | |
MgSO4 | Sigma-Aldrich | M7506-500G | |
NaCl | Sigma-Aldrich | S9888-25G | |
NaHCO3 | Sigma-Aldrich | S6014-25G | |
Roller Pump | Gilson | Minipuls 3 | |
straight dissecting scissors | Kent | INS600393-G | |
Temporary cardiac pacing wire | Ethicon | TPW30 | |
Wide Range Force Transducer | ADInstruments | MLT1030/A |