This research aims to highlight the dynamism and broad application of Langendorff perfusion as a research tool and its immense utility in cardiovascular disease research. It also highlights the importance of tailoring the different perfusion parameters, such as flows, pressures, perfusate, and temperature, to better fit experimental needs. This protocol offers an alternative approach to the current status quo of Langendorff perfusion.
It suggests that using lower-than-normal perfusion pressures, such as 30 to 35 millimeters of mercury instead of the usual 60 to 80 millimeters of mercury, could be beneficial for certain types of experiments and could improve overall scientific outcomes of relevant studies. This protocol enhances the significance of Langendorff perfusion as a technique for the cardiovascular research by eliminating the loss of function that is dependent on the technique. Furthermore, it is expected to start a conversation about the best perfusion practices for clinically perfused cardiac grafts, such as those that are being preserved for transplantation.
To begin, remove the anesthetized rat from the anesthetic chamber once reflexes are no longer observed. Place it into the surgical space and deliver continuous isoflurane at 3%via face mask. After conducting a toe pinch test, administer 30 units of heparin through the penile vein of the animal.
Shave the rat across the entire abdomen and upper chest area. Then remove the fur shavings from the surgical field. Secure each limb with tape to prevent movement during surgery.
Now make a horizontal midline incision in the skin of the lower abdomen to expose the abdominal muscles. Then make a second horizontal midline incision through the abdominal muscles to expose the internal organs. Next, expose the sternum.
Secure it with a hemostat and retract it cranially to display the liver and portal vein. Using a 16-gauge angio catheter, insert a catheter into the portal vein and connect a 60-milliliter syringe of heparinized saline to the angio catheter. Then create an incision in the inferior vena cava and abdominal aorta for venting.
Flush the entire amount of saline through the portal vein. Afterward, make a horizontal cut in the diaphragm, followed by a proximal cut through the ribs on both sides of the sternum to reveal the thoracic cavity. Remove the heart from the cavity and immediately place it on a Petri dish with saline on ice.
Then identify the aortic arch, clamp it with hemostats, and expose the descending aorta by clearing any remaining connective tissue. Using dissecting micro scissors, make a horizontal cut halfway across the descending aorta and insert a 14-gauge angio catheter. Secure the cannula with a suture above the cuff and release the hemostat.
To begin, set the peristaltic pump's flow to 1.0 milliliters per minute. Weigh the isolated rat's heart and cannula before attaching them to the system. Connect the cannula to the system's connector and start a timer.
Once the heart contracts fully, increase the flow in 0.2 milliliter per minute increments while monitoring the pressures. Stop increasing the flow when the desired pressures are reached, or when a minimum flow of 3.5 milliliters per minute is achieved. For pressures between 30 and 35 millimeters of mercury, set the flow to 4.5 milliliters per minute.
Then start the adenosine syringe pump. Using a Luer-lock connector, connect the catheter to a pressure sensor and secure the entire setup to a clamp stand. After that, attach a small latex balloon to a balloon catheter with a tapered tip.
Using a syringe attached to the top end of the pressure sensor, fill the balloon catheter and pressure sensor with approximately 200 microliters of saline. Then, with the help of a sphygmomanometer, calibrate the pressure sensor. Next, make a small horizontal incision above the left atrium.
Deflate the balloon by drawing back the syringe at the top of the pressure sensor and inserting it into the left ventricle. Afterward, start data acquisition and inflate the balloon until the diastolic pressures indicate zero millimeters of mercury. A clear correlation was established between heart rate and perfusion pressures.
Notably, heart rate was significantly higher in high-pressure hearts compared to low-pressure hearts throughout the study with the exception of the initial measurement. There was a significant disparity in left ventricular pulse pressure or LVPP between the groups, with high-pressure hearts displaying statistically higher LVPP across all time points. Over time, high-pressure hearts exhibited a progressive decline in function, with a noticeable decrease in LVPP after two hours.
Conversely, low-pressure hearts maintained consistent LVPP throughout the perfusion. High-pressure hearts demonstrated superior cardiac function compared to low-pressure hearts.
