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.
