The overall goal of this procedure is to model ischemia, reperfusion injury, ex vivo. This is accomplished by first anesthetizing, a rat, and opening up the chest to cannulate the aorta in C two, and thus prevent ischemic preconditioning of the myocardium. Next, the heart is removed and attached to alanor perfusion apparatus.
The final step is to administer an ischemia reperfusion injury by stopping the buffer flow for a period, then restoring flow. Ultimately, ventricular pressure generation is used to show the effect of ischemia reperfusion injury on myocardial function. This method can be used to help answer key questions in the cardiac ischemia reperfusion field, such as what molecular events occur during the dynamic first few hours of ischemia reperfusion injury.
To begin, calculate the required anesthetic after the rat has been confirmed to be anesthetized. Begin the tracheotomy by first opening the skin from the mid four paw to the base of the jaw. Use hemostats to place it aside, exposing the musculature.
Then with hemostats, lift the glandular tissue and use scissors to make an inferior incision into the fascia. Spread apart the exposed tissue with another pair of hemostats. And be careful not to nick the jugular.
Now make a transverse incision in the muscle overlying the trachea with scissors and the aid of hemostats. Then use the hemostats to reveal the trachea carefully position hemostats under the trachea, clearing the fascia in the process. Then pinch zero, zero silk suture with the hemostats behind the trachea.
Next, use scissors to hemis. Dissect the trachea into the trachea. Insert the tracheal cannula and secure it with the suture, not the suture to the Y groin of the cannula.
Now from a syringe with a 27 gauge needle, inject 1000 units of heparin per kilogram into the jugular vein. Wait 30 seconds before proceeding with the thoracotomy. Begin the thoracotomy by removing the skin between the mid abdomen and mid four pa using hemostats and scissors.
Then make a three quarter inch incision into the abdominal wall just below the diaphragm. Continue the incision vertically along the sternum. Then cut the diaphragm bilaterally to expose the rib cage.
Now, spread the rib cage and clamp it aside with hemostats, thus revealing the heart. Situ two cannulation of the aorta is the most critical step of the procedure. The cannula must be inserted accurately and rapidly tied off in order to prevent the heart from experiencing a brief period of ischemia, which would alter the results of the subsequent ischemia reperfusion.
Experiment With hemostats. Carefully remove the thymus to access the ascending aorta. Maneuver hemostats through the aortic loop through the fascia.
Then feed zero zero silk suture through the opening and tie in a loose overhand knot. Now start the profusion buffer flow through the cannula. Next hemis, dissect the aorta and immediately insert the cannula into the aortic lumen.
Secure the cannula by cinching the knot and completing it into a square knot. Using scissors, dissect the heart away from the great vessels. Once freed, remove it and attach it to the profusion column.
Trim away the excess lung tissue. After about 15 minutes, the heart will be equilibrated with the normal buffer flow of between 10 and 20 milliliters per minute. Once the heart is equated to the column, the balloon can be inserted.
First, deflate the balloon, rolling it into a cone shape. Lock the flow to the balloon once it is deflated. Second, access the mitral valve by excising the left atrium of the heart.
Third, insert the balloon via the mitral valve into the left ventricle. Now start monitoring systolic and diastolic pressure with lab chart pro software inflate the balloon until the pressure is greater than zero. Then check the length tension response.
Inflate the balloon slowly until the pressure reaches 75 millimeters of mercury. Then deflate it slowly and repeat. Lower the pressure back down to 10 millimeters by altering the saline in the balloon.
Then lock the balloon at this pressure and maintain a closed pressure system between the balloon and transducer. Lastly, lower the heart into a heated chamber and close it in plastic wrap. The chamber should be plugged so it fills with a fused buffer.
Once the heart is submerged, stop the buffer flow. This induces global ischemia for as long as needed. In this case, 30 minutes.
After 30 minutes, stop the ischemia by restoring buffer flow into the heart and unplugging the chamber. Allow the reperfusion to go as long as desired and then terminate the experiment, which in this case will be after an hour of reperfusion from the side port of the stop cock. Administer 10 milliliters of ice cold PBS over one minute.
This will arrest the heart. Then remove the heart from the column and trim away any remaining atrial tissue. Pat the heart dry, transfer it to a slicing matrix and cover it in perfil.
Freeze the heart at minus 80 degrees Celsius for eight minutes or until it has the consistency of a marshmallow. Using razor blades, section the heart into two millimeter sections, or as preferred, remove each slice individually and freeze it in liquid nitrogen. For biochemical studies, the frozen slices should then be stored at minus 80 degrees Celsius.
For infarct staining retain one slice unfrozen in liquid nitrogen. With the described protocol. The left ventricular balloon apparatus allows for real-time monitoring of the pressure developed by the contracting left ventricle.
In a normal heart, the developed pressure ranges from 70 to 130 millimeters of mercury. After an ischemic insult, the developed pressure is reduced and the end diastolic pressure elevated when animals are administered. A preconditioning agent like Class one and two B HDAC inhibitor saha prior to excision, baseline pressure may vary and the effects of ischemia reperfusion injury are attenuated.
Other measures of left ventricular function can also be directly obtained or calculated from the software output. Upon investigating the injured heart with TTC, which stains metabolically active tissue red, it was evident that the heart subjected to ischemia reperfusion injury were substantially less metabolic, indicating that they contained infarcted tissue Following this procedure. Techniques such as real-time quantitative PCR, Western blotting and mass spectrometry can be used to answer questions such as, what are the biochemical and genetic effects of ischemia, reperfusion injury, and drug treatment on the heart?
