Name: modified technique for the use of neonatal murine hearts in the langendorff preparation
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NIH/NINDS R01NS112706 (R.L.)

Institutional Animal Care and Use Committee of Columbia University Medical Center&#39;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
<ol>
	<li>To minimize complexity, use non-recirculating oxygenated perfusate within the Langendorff apparatus (see Table of Materials) via constant flow or constant pressure.
	<ol>
		<li>Use Krebs-Hensele...

<ol>
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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...

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 &#34;working heart&#34;) 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&#39;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&#39;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 &#39;working heart&#39; 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 &#34;relatively immature&#34; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rodent Langendorff Apparatus</td>
			<td>Radnoti</td>
			<td>130102EZ</td>
			<td></td>
		</tr>
		<tr>
			<td>24 G catheter</td>
			<td>BD</td>
			<td>381511</td>
			<td></td>
		</tr>
		<tr>
			<td>26 G needle on 1 mL syringe combo</td>
			<td>BD</td>
			<td>309597</td>
			<td></td>
		</tr>
		<tr>
			<td>26 G steel needle</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Silk Suture</td>
			<td>Ethicon</td>
			<td>S1173</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio Amp</td>
			<td>ADInstruments</td>
			<td>FE135</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio Cable</td>
			<td>ADInstruments</td>
			<td>MLA1515</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C4901-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Circulating heating water Bath</td>
			<td>Haake</td>
			<td>DC10</td>
			<td></td>
		</tr>
		<tr>
			<td>curved iris scissor</td>
			<td>Medline</td>
			<td>MDS10033Z</td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>find spring scissors</td>
			<td>Kent</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr>
			<td>Force Transducer</td>
			<td>ADInstruments</td>
			<td>MLT1030/D</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent</td>
			<td>400-01</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure tubing</td>
			<td>Edwards Lifesciences</td>
			<td>50P184</td>
			<td></td>
		</tr>
		<tr>
			<td>iris dressing forceps</td>
			<td>Kent</td>
			<td>INS650915-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Jeweler-style curved fine forceps</td>
			<td>Miltex</td>
			<td>17-307-MLTX</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P3911-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P0662-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M7506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6014-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Gilson</td>
			<td>Minipuls 3</td>
			<td></td>
		</tr>
		<tr>
			<td>straight dissecting scissors</td>
			<td>Kent</td>
			<td>INS600393-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Temporary cardiac pacing wire</td>
			<td>Ethicon</td>
			<td>TPW30</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Range Force Transducer</td>
			<td>ADInstruments</td>
			<td>MLT1030/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

modified technique for the use of neonatal murine hearts in the langendorff preparation

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.

P10 mice were used to model&#160;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 ele...

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

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 ...

This approach reliably establishes retrograde perfusion of the neonatal ex vivo mouse heart in under three minutes. Thus reducing the youngest possible age of study from several weeks to 10 postnatal days. This isolated retrograde perfused model allows for the study of the neonatal heart during a critical period of cardiac development in a genetically modifiable and low cost species.While this model lends itself to the study of physiological and pharmacological responses to ischaemia-reperfusion in the neonatal heart. It could also be utilized for cardiomyocyte isolation. To fabricate the newborn mouse aortic cannula use sharp scissors to cut off the tip and blunt the end of a 26 gauge stainless steel needle, taking care not to crimp or restrict the diameter of the needle lumen.Then smoothen the cut edge to remove any burrs by gently scraping the blunted end on the laboratory bench top using a to-and-fro motion. Next, attach the fabricated cannula to the Langendorff apparatus and assess flow and resistance. Measure flow rates through the cannula by collecting and measuring the buffer quantity over a one minute interval.To quantify the pressure differential across the cannula while the Krebs-Heneseleit buffer is flowing, measure the pressure in the system with and without the cannula attached. Then calculate the cannula resistance using the following formula. Next, remove the 26 gauge cannula from the Langendorff apparatus and attach a high pressure tubing to the cannulation site on the apparatus.Then attach the aortic cannula to the distal end of the tubing and de air the tubing and the cannula with oxygenated buffer, ensuring that all bubbles are removed. To prevent the formation of coronary microthrombi, administer an intraperitoneal injection of heparin to a 10 day old mouse pup using a 26 gauge needle on a one milliliter syringe. Allow several minutes for the heparin to circulate before proceeding with the injection of any anesthetic.Next, place the anesthetized mouse in the supine position and secure limbs immediately upon loss of consciousness. Begin harvesting as soon as the animal is unresponsive to toe pinch as the animal breathes spontaneously during the initial dissection. Using straight dissecting scissors make a transverse subxiphoid incision across the animals width exposing the abdominal cavity.Ask an assistant to grasp the xiphoid process with forceps, then cut the ribcage bilaterally along the mid axillary line in cephalad direction and reflect the sternum and ribs cranially to expose the thoracic organs. After identifying the diaphragm superiorly, incise the anterior portion completely. Identify the infradiaphragmatic inferior vena cava above the liver and transect it with a curved Iris scissor while maintaining slight interior in cephalad tension on the proximal segment with Iris forceps.Then, cut posteriorly along the interior surface of the spine using curved Iris scissors while pulling the inferior vena cava 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. Immediately submerge the specimen in ice cold Krebs-Henseleit buffer or saline.The heart should stop beating within seconds. Place a piece of paper towel at the bottom of a shallow Petri dish to provide friction to stabilize the heart during cannulation. Moisten the paper towel with ice cold Krebs-Henseleit buffer to prevent the heart from adhering to it.Place the prepared Petri dish under the dissecting microscope and adjust the focus. Now, 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. After placing the excised thoracic organs in the Petri dish, identify the thymus by its white sheen and two lobes and orient the specimen such that the thymus is anterior and superior.Next, using forceps bluntly separate the lobes of the thymus exposing the great vessels. Then identify the aorta by locating distinguishing branching features of the aortic arch and transect it just proximal to the subclavian artery takeoff using fine sharp scissors. Now gently grasp the transected aorta using jeweler style fine curved forceps and carefully cannulate the aorta with a 26 gauge blunt needle, taking care not to damage the aortic valve.Using fine curved forceps, grasp the aorta around the cannula and initiate retrograde perfusion to limit the ischemic time. 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 fine curved forceps then tighten the suture and confirm the adequacy of coronary flow.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. Then rapidly attach the hub of the cannula to the apparatus. Once the heart is hung on the Langendorff apparatus in the usual position, an adequate perfusion is confirmed.Carefully trim off the lung, thymus and excess tissue. Then incise the right atrium to permit the coronary sinus effluent to drip freely. Make a small knot at the end of a 5-0 silk suture, then pierce a small piece of paraffin film 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. Pass the needle through the opening of the water filled warming jacket of the Langendorff apparatus so that the heart can be encased and warmed. Then attach the needle to the force transducer avoiding the coronary sinus drip.Adjust the suture to apply one to two grams of basal tension as indicated by the diastolic tension or nadir intention tracing. Then place surface electrodes on the superior and inferior poles of the heart to record the electrocardiogram. Finally, sample the coronary sinus effluent for analysis using a 24 gauge intravenous catheter.Surface electrodes were used to record a continuous electrocardiogram, which allowed determining intrinsic rate and rhythm. All the adequately perfused hearts beats spontaneously in sinus rhythm. 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.Physiologic variable means, like the mean denervated intrinsic heart rate and mean observed aortic perfusion pressures were also recorded and calculated. Some exclusionary criteria such as the time required to initiate reperfusion, arrhythmia duration and aortic perfusion pressure must be considered to ensure consistency of the neonatal preparation. After establishment of perfusion, intervention such as drug delivery, induction of ischemia-reperfusion injury, alterations of metabolites or delivery of enzymatic agents for cardiomyocyte isolation may occur.

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