Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation

Summary

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.

Abstract

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.

Introduction

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 recently^8,9. 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 availability^12,13. 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 heart^11,14. 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.

Protocol

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

1. To minimize complexity, use non-recirculating oxygenated perfusate within the Langendorff apparatus (see Table of Materials) via constant flow or constant pressure.
   1. Use Krebs-Henseleit buffer (KHB), containing 120 mmol/L of NaCl, 4.7 mmol/L of KCl, 1.2 mmol/L of MgSO₄, 1.2 mmol/L of KH₂PO₄, 1.25 mmol/L of CaCl₂, 25 mmol/L of NaHCO₃, and 11 mmol/L of glucose at pH 7.4 (see Table of Materials), equilibrate with 95% of O₂ and 5% of CO₂ within the Langendorff apparatus and maintain at 37 °C.
2. For the constant flow approach, maintain a continuous flow rate at ~2.5 mL∙min^-1.
   NOTE: This flow rate will approximate coronary flow of ~75-80 mL/g∙min, given that the average weight of a 10 day old (P10) mouse heart is ~30 mg^17,18.

2. Fabrication of aortic cannula

1. Fabricate the newborn mouse aortic cannula from a 26 G stainless steel needle (see Table of Materials). Using sharp scissors, cut off the tip of the needle to blunt the end. Take care not to crimp or restrict the diameter of the needle lumen. Smooth the cut edge and remove any burs by gently scraping the blunted end on the laboratory benchtop using a to-and-fro motion.
   NOTE: Microscopic burs and sharp edges must be removed because they can tear the newborn mouse aorta and damage the aortic valve. Alternatively, use fine-grit sandpaper.
2. Attach the fabricated cannula to the Langendorff apparatus and assess flow and resistance. Measure flow rates through the cannula by collecting and measuring buffer quantity over a known time period. Ensure actual flow is equal to the set flow rate of 2.5 mL min^-1.
3. Quantify the pressure differential across the cannula with KHB flowing by following the steps below.
   1. Measure pressure in the system with and without the fabricated cannula attached.
   2. Divide pressure differential across cannula by the flow rate to obtain cannula resistance as per Ohm's law¹⁵.
   3. Ensure that the fabricated cannula resistance comprises ~16.0 ± 1.9 mmHg∙min∙mL^-1 of the total resistance⁶. Excessive resistance suggests a potentially compromised cannula lumen.
      NOTE: Sample calculation: P_{with cannula} - P _{without cannula} = ΔP. If P_with = 48 and P_without = 8 then ΔP = 40. At a flow rate (Q) of 2.5 mL min^-1 and ΔP of 40 cannula resistance equals 16 mmHg∙min∙mL^-1 using R = ΔP/Q = 40 / 2.5 = 16.
4. Remove the 26 G cannula and attach the high-pressure tubing (see Table of Materials) to the cannulation site on the Langendorff apparatus. Attach the aortic cannula to the distal end of the tubing. De-air the tubing and the cannula with oxygenated buffer, ensuring that all bubbles are removed.
   NOTE: The use of high-pressure tubing in this manner permits the cannula to be extended to a more remote position. This is necessary to allow aortic cannulation with a dissecting microscope adjacent to the setup (Figure 1).

3. Organ harvesting

1. Anticoagulate mice via intraperitoneal (IP) injection of heparin (10 kU/kg) (see Table of Materials) to prevent the formation of coronary microthrombi using a 26 G needle on 1 mL syringe. Allow several minutes for heparin to circulate before proceeding with the injection of any anesthetic.
2. Anesthetize the animal with an IP injection using a 26 G needle on 1 mL syringe.
   NOTE: It is essential to carefully monitor the animal after anesthetic injection to avoid apnea and subsequent hypoxia. Pentobarbital (70 mg/kg) is a reliable choice of anesthetic, as it allows for rapid onset of sedation without inducing apnea^19,20. Other anesthetic agents can be utilized, provided that the doses used do not cause apnea²¹. Investigators should consider the effects of alternative sedative-hypnotics on cardiac function^22,23. Cervical dislocation as a primary mode of euthanasia may prolong pre-cannulation hypoxia and ischemia.
3. Place the mouse in the supine position and secure limbs immediately upon loss of consciousness. Use small gauge hypodermic needles to secure each limb. Begin harvesting as soon as the animal is unresponsive to toe pinch; the animal should breathe spontaneously during the initial dissection.
4. Make a transverse subxiphoid incision across the animal's width to expose the abdominal cavity using straight dissecting scissors (see Table of Materials).
   NOTE: Sterile technique is not necessary given that the procedure represents nonsurvival surgery.
   1. Identify the diaphragm superiorly and incise the anterior portion completely. Cut the ribcage bilaterally along the mid-axillary line in a cephalad direction. Ask an assistant to grasp the xiphoid process with forceps and reflect the sternum and ribs cranially to expose the thoracic organs.
5. Identify the infra-diaphragmatic inferior vena cava (IVC) above the liver. Transect the IVC with a curved iris scissor while maintaining slight anterior and cephalad tension on the proximal segment with iris forceps (see Table of Materials).
   1. Cut posteriorly along the anterior surface of the spine using curved iris scissors while pulling the IVC up and out of the thoracic cavity. As the heart is mobilized, angle the scissors anteriorly and sever the great vessels superiorly to completely remove the heart and lungs.
      ​NOTE: This method permits rapid explantation of the heart and lungs en bloc.
6. Immediately submerge the specimen in ice-cold KHB or saline. The heart should stop beating within seconds.

4. Cannulation

1. Cut a piece of paper towel and place it at the bottom of a shallow Petri dish to provide friction to stabilize the heart during cannulation. Moisten with ice-cold KHB to prevent the heart from adhering to it.
   1. Place the prepared Petri dish under the dissecting microscope and adjust the focus. Place the aortic cannula attached to the high-pressure extension tubing under the dissecting microscope along with a 5-0 silk suture loosely tied around its hub (see Table of Materials).
      NOTE: Care must be taken to limit the amount of fluid in the Petri dish because the air-filled lungs can float and cause the excised organs to move.
2. Place the excised thoracic organs in the Petri dish. Under the microscope, identify the thymus by its white sheen and two lobes and orient the specimen such that the thymus is anterior and superior²⁴. This will ensure proper orientation of the heart.
3. Using forceps, bluntly separate the lobes of the thymus to expose the great vessels. Identify the aorta by locating distinguishing branching features of the aortic arch.
   NOTE: A dark purple hue often demarcates the right ventricle and the pulmonary artery. The ascending aorta is located between the main pulmonary artery and the right atrium.
4. Transect the aorta with fine sharp scissors (see Table of Materials) just proximal to the subclavian artery takeoff.
   NOTE: If the aorta is transected too close to the aortic valve, there will not be enough aortic tissue to enable the cannula to be secured. Alternatively, if the aorta is transected too high, perfusate can leak out of one or more aortic branches (such as the subclavian artery).
5. Gently grasp the transected aorta using jeweler-style fine curved forceps (see Table of Materials). Carefully cannulate the aorta with a 26 G blunt needle, taking care not to damage the aortic valve. Hold in place by grasping the aorta with the fine curved forceps around the cannula. Once control of the aorta is established, initiate retrograde perfusion to limit the ischemic time.
   NOTE: The heart should begin to beat and will become pale as blood is drained from the myocardium and KHB perfuses the coronary arteries. Failure to spontaneously beat, presence of ventricular engorgement, or lack of color change of the heart indicates a malpositioned cannula.
6. Ask the assistant to grasp the ends of the loosely tied suture and carefully ensnare the aorta around the cannula. Cinch the suture above or below the curved fine forceps (holding the cannula in place), depending on the amount of aortic tissue and anatomical considerations. Tighten the suture and confirm the adequacy of coronary flow.
7. Disconnect the high-pressure tubing from the Langendorff apparatus. Grasp the hub of the cannula and disconnect the blunt needle from the high-pressure extension tubing. Rapidly attach the hub of the cannula to the apparatus.
   NOTE: Care must be taken not to dislodge the heart or entrain air into the cannula.
8. Once the heart is hung on the Langendorff apparatus in the usual position, and adequate perfusion is confirmed, carefully trim off lung, thymus, and excess tissue. Incise the right atrium to permit coronary sinus effluent to drip freely.

5. Functional measurement

1. Make a small knot at the end of a 5-0 silk suture (attached to a curved needle). Pierce a small piece of paraffin film (2-3 mm x 2-3 mm) with the needle and slide the paraffin to the knotted end. Carefully pass the needle through the apex of the ventricle and pull the suture through the heart until the paraffin film is snug against the lateral wall of the ventricle.
   NOTE: The paraffin film helps to prevent the knot from tearing the heart and pulling through the ventricle.
2. Pass the needle through the opening of the water-filled warming jacket of the Langendorff apparatus. The heart can now be encased and warmed.
3. Attach the needle to the force transducer (see Table of Materials) in such a manner that avoids the coronary sinus drip. Adjust the suture to apply 1-2 g of basal tension, as indicated by the diastolic tension or nadir in tension tracing.
   NOTE: Avoid pulling the heart off the cannula or twisting the aorta, thereby compromising coronary perfusion.
4. Place surface electrodes on the superior and inferior poles of the heart to record the electrocardiogram.
   NOTE: Use pediatric temporary epicardial pacing wire with the needle removed for flexible surface electrode connected to Bio Amp (see Table of Materials).
5. Sample the coronary sinus effluent for analysis using a 24 G IV catheter (see Table of Materials).
6. Subtract the cannula resistance from the total system resistance to obtain coronary resistance per Kirchhoff's law²⁵.

Results

P10 mice were used to model a timepoint in human infancy^26,27. 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 electrod...

Discussion

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. D...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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