This protocol describes a method to collect cardiac interstitial fluid from the isolated, perfused rat heart. To physically separate interstitial transudate from coronary venous effluent perfusate, the Langendorff perfused heart is inverted, and the transudate (interstitial fluid) formed on the cardiac surface is collected using a soft latex cap.
The overall goal of this inverted heart model is to collect cardiac interstitial transudate from an isolate rat heart. Within the beating heart, the individual sense of the heart release paracrine factors and they release those into the surrounding interstitial fluid space. This is important, because the interstitial fluid space, it carries information for the communication between the cardiac cells.
And that is particularly important under conditions of inflammation and regeneration of the infarcted myocardium. In this video, my collaborators, Dr.Ding and Tan, will show you the techniques by which we here, in this uh-love collect interstitial fluid from the surface of an isolated perfused rat heart. To make the latex heart cover, or cap, have an aluminum mold that matches the average size of a rat's heart and has been polished with superfine emery paper.
Vertically fix the neck of the mold to the milling machine and start to slowly rotate the mold. Then, pour 20 milliliters of liquid latex into a 50 milliliter glass beaker, and lower the rotating mold into the latex until it is immersed. Then, slowly lift it out and keep rotating the mold for an additional 15 minutes until the latex has solidified.
The rotation is vital to make this mousse and evenly distribute layer of the liquid latex on the mold's surface. So, make certain that the mold is rotating as it is lifted from the latex. Once the latex is dry, coat the latex with talcum powder to prevent damage to the latex.
Stop the rotation, then gently detach the mold from the machine. Carefully remove the latex from the mold. Now, attach a 25 centimeter length of silicon tubing to the outlet on the latex cap.
This tube is used to collect the interstitial cardiac transudate, or CT.Next, firmly attach the rat-sized ventricular balloon onto an L-shaped metal cannula, connected to a one milliliter water-filled syringe. And fill the rat-sized ventricular balloon with water. Make sure that the balloon is airtight by inflating and deflating it several times using the syringe.
Then, connect the cannula to a pressure transducer, via a three way stop. Lastly, make a mark on the syringe where the balloon will fill the ventricular cavity for the measurement of intraventricular pressure. Before proceeding with preparation of the heart, ready the Langendorff perfusion system according to the text protocol.
Now, after fully anesthetizing a 300 to 350 gram male Wistar rat, transfer it to a room temperature operating table. With experience, the following steps leading to the cannulation of the heart should only take about three minutes. Lift the skin and muscle just below the sternum, using forceps, and cut around the lower margin of the ribs using heavy scissors.
Next, with fine scissors, make a small cut into the diaphragm at the rib margin. Next, cut the rib's caudally to make a flap of the entire ventral chest wall. Now, gently take hold of the heart, and slowly life it until the cardiac vessels start to stretch.
Then, excise the heart until the aorta is fully exposed and transfer the heart to a 100 milliliter beaker containing 50 milliliters of ice cold KHB. Now, immediately take the heart to the profusion apparatus, and mount the aorta onto a dripping cannula. Secure it tightly with a 4-0 suture, so that no air bubbles can enter the heart.
Then, apply either a constant profusion pressure or a profusion flow. To establish the reversed heart set up, first gently rotate the aortic cannula until the posterior wall of the hart is en face. Then, snip away the connective tissue to expose the opening to the left atrium.
Next, gently grasp the heart and turn it upside-down. Mount the heart onto the prepared ventricular balloon, with the balloon deflated, and feed it into the left ventricle. The heart is now mechanically stabilized in an upright position, using the balloon catheter assembly as a support.
Inflate the balloon until it fills the entire ventricular cavity, by advancing the plunger to the fill mark. Secure the heart so there is minimal stress on the aortic root. This part of the procedure takes practice.
Now, observe the gauge on the balloon, and adjust the diastolic pressure to three to five millimeters of mercury. Small droplets should form on the epicardium if the pressure is set correctly. Next, gently pull the latex cap over the heart.
Even the most ventricular surface should be covered. Before proceeding, check for air bubbles inside the cap. To remove any air bubbles, use gentle suction from the syringe.
Now, position the end of the letting tube 10 centimeters below the heart. Collect the dripping CT into a 1.5 milliliter tube and keep the tube below four degrees Celsius by embedding it in a 1:1 mixture of ice and sodium chloride. Collect between 0.15 and 1.5 milliliters of CT, then weight the sample and snap freeze it in liquid nitrogen.
Store it at minus 80 degrees Celsius until it can be processed. When retroperfusing the reversed heart model at a constant pressure of 100 centimeters of water, between 1/10 to 1%of the coronary venous effluent could be collected. The protein content of the CT was measured with the BCA assay, and averaged 1.1 milligram per milliliter.
Simple gel electrophoresis revealed a wide spectrum of proteins. Normal samples were compared to samples from hearts subjected to 50 minutes of ischemia reperfusion using 2D DIGE. Several protein were found to be upregulated in the CT of the ischemic hearts.
Proteomic analysis was performed on the upregulated proteins. Purines were also of great interest. Purines are pivotal signaling molecules that regulate the cardiac immune response, vasomotor tone, and cardiac function, particularly after ischemic injury.
HPLC analysis of the CT showed elevated concentrations of many purines in the injured hearts. While attempting this procedure, it's important to remember that a cardiac trans-in-date being formed in an isolated heart, will not entirely mimic the true composition of the in-vee-vuh conditions.
