The overall goal of this procedure is to assess the myocardial stunning that occurs following ischemic cardioplegic arrest in rodents. This is accomplished by first retrograde perfusing, a heart harvested from an adult mouse. In the second step, a balloon is inserted into the left ventricle of the heart and the baseline control measurements are recorded.
Hypothermic cardioplegic arrest is then initiated for the appropriate experimental period followed by a period of reperfusion. Ultimately, this technique can be used to monitor the changes that occur during the recovery of myocardial function after ischemic injury. Though this method can provide insight into myocardial stunning associated with mild ischemic insults in the mouse can also be applied to other mammals, such as rats and two models of severe ischemia reperfusion injury.
Generally, individuals new to this method will struggle because it can be difficult to quickly cannulate the heart, a requirement for a stable and reproducible preparation. To remove the heart, a thoracotomy is performed. The first step is to cut a small hole just below the sternum, extending the incision to the sides of the mouse.
Then quickly cut out the diaphragm and rapidly up the sides of the ribcage. Flip the ribcage back like a clamshell to expose the thoracic cavity. Then gently take hold of the heart, place the scissors underneath and remove the organ.
Place the heart into a dish of ice cold crebs henzel light buffer, or KHB, and trim off any large pieces of lung tissue. Then gently squeeze the heart. Any blood that appears should indicate the open end of the aorta.
Before attaching the heart, turn on the pump and confirm that the perfusion pressure is approximately 20 millimeters of mercury. Then use fine tweezers to pick up the heart by the open end of the aorta and use another set of fine tweezers to slide the heart up onto the perfusion system cannula, holding the heart in place. Use a soft edged clip to temporarily secure the aorta to the cannula.
Next, use a four zero silk suture to secure the aorta to the cannula directly below the clip. Taking care to place the tie around the cannula also so that the aorta does not get tied off when tightening the knot. Once the tie is firmly in place, remove the clip and use additional silk sutures to fasten the aorta securely to the cannula.
Tying the sutures off below any vessel branches coming off of the aorta that are leaking or shooting out perfuse eight. During the initial stages of the perfusion, the blood should wash out from the heart and the entire organ should turn a soft pink color When the cannula has been secured, slowly increase the profusion pressure and pump speed until the profusion pressure reaches 70 millimeters of mercury. Remove any remaining tissue still attached to the heart.
Then cut off the left atria to create an opening in the left ventricle. Now place a balloon onto the holder and deflate it. Then position the balloon cannula near the aortic cannula directly over the opening into the left ventricle, and carefully insert the balloon downward into the left ventricle while holding the heart in place so that the aorta does not tear.
Once the balloon is in place, slowly start to inflate the balloon until the left ventricular end diastolic pressure reaches approximately eight millimeters of mercury. Then place a temperature probe against the bottom of the heart, so as to measure the temperature of the effluent. To take the basal measurement seal the heart in a water jacketed perfusion chamber set to 37 degrees Celsius with continuous KHB perfusion.
Then begin the continuous recording of the functional measurements of interest using the appropriate data acquisition system. Once the effluent reaches approximately 37 degrees Celsius for at least 15 minutes, and the cardiac functional parameters are stable, note the time of the baseline measurements. Then measure the coronary flow via the collection of the coronary effluent and a graduated cylinder.
After one minute, close the chamber and allow the temperature and function to return to baseline values. To initiate the arrest now, add 100 milliliters of cold cardioplegic or CP buffer into a second reservoir and transfer the KHB reservoir cannula to the CP reservoir. Using quick tubing disconnects, transfer the water jacketed housing and the profusion warming circuit From the warm water bath to the refrigerated circulator, follow the air bubble that gets introduced into the profusion tubing during the switch.
Once it enters the bubble trap near the isolated heart, start the timing for the cardioplegia and deliver the buffer for two minutes. As the CP is delivered and the heart cools, cardiac function will decrease and it will arrest diastole After the initial dose. Stop the pump and keep the heart in the CP buffer for two hours at approximately 20 degrees Celsius, turning the pump on every half hour for one minute to administer another dose at the end of the CPE period, transfer the reservoir cannula back to the oxygenated KHB and connect the temperature control circuits to the heating circulator.
Then turn on the pump to start a 30 minute reperfusion washing out the CP buffer. After two to four minutes, the temperature will rise and the heart will begin to beat slowly and a rhythmically collect the effluent in a graduated cylinder for 30 to 60 seconds at a time to obtain the appropriate coronary flow measurements. Here, typical results from one mouse experiment are shown the left ventricular pressure positive and negative first derivative of the left ventricular pressure or LVP and temperature are continuously recorded over approximately three hours as just demonstrated upon reperfusion a depressed LVP and a depressed positive and negative first derivative of the LVP compared to baseline were observed here.
The perfusion pressure LVP and positive and negative first derivative of the LVP data from the previous figure recorded over two seconds before and after the ischemic CP arrest are shown. Demonstrating how changes in the LVP and the positive and negative first derivative of the LVP data are observed After the induction of cardioplegia after 30 minutes of reperfusion and approximate 30 to 40%decrease in cardiac function is typically observed as evidenced by the left ventricular developed pressure data tracked in this last graph. Once mastered, the heart can be harvested and mounted onto the perfusion apparatus in approximately one to two minutes if it is properly performed.
After watching this video, you should have a good understanding of how to remove and lor perfuse a mouse heart, the subsequent hypothermic cardioplegic arrest and reperfusion.
