With the described protocol, we can compare the donor heart left ventricle, and ventricle function after preservation, and we can examine the difference of preservation biology in both ventricles during these preservation process. This technique use ex vivo perfusion system to examine the heart functions after preservation. It provides a robust and reliable tools to screen new treatments to improve donor heart functions after preservation.
Begin with the preparation of dough as described in the text and shape a small piece of the dough into an oval shape to obtain the head. Attach it to the end of a dry spaghetti strand. Dip the head into the already prepared sugar solution and slowly remove it once it is well-coated with a thin film of the solution.
Then, suspend the spaghetti strand on a polystyrene foam block or other holders to allow the formation of an even and glossy cover over the head and dry it overnight. The next day, dip the mold into silicone elastomer dispersed in xylene and place the spaghetti strand back into the polystyrene foam block at 37 degrees Celsius for two hours or until dry. Repeat this step one more time.
Then, place the mold into the water to separate and collect the balloon. Store the balloon in 0.02%sodium azide. Now, cut a two blunt end tip from a 22-gauge needle and mount one blunt end to the silicone balloon while the other blunt end to a PE tubing.
Use a 4-0 silk suture to tie the balloon in place on the needle. Take the C57Black/6 mouse and intraperitoneally inject 200 units of heparin into the animal's abdomen for anticoagulation. After confirming proper anesthesia with a toe pinch, make an incision right below the sternum.
And using scissors, open the chest by cutting the diaphragm in the ribs. Then, fold the anterior chest wall to fully expose the chest before making a cut at the descending aorta close to the aorta arch. Now, transfer the mouse's heart, lungs, and thymus to ice-cold histidine-tryptophan-ketoglutarate, or HTK buffer, to isolate the organs.
Expose the aorta by removing any connective tissue. Connect the end of the aorta to a 22-gauge needle and tie it with a 6-0 silk suture. Ensure the cannula is above the aortic root, so as not to interfere with the aortic valve.
Perfuse the aorta for approximately 10 minutes with 10 milliliters of HTK buffer, pre-cooled to four degrees Celsius. Depending on the type of experiment, either store the heart in a 50-milliliter tube with ice-cold HTK for eight hours or immediately perform the profusion. To perform profusion, connect the needle-mounted heart to the cannula in the Langendorff apparatus and tie it with a silk suture.
Start the profusion in a constant flow mode at three milliliters per minute, and then after changing to a constant pressure mode at 70 to 80 millimeters of mercury, adjust the perfusion rate to approximately six milliliters per minute. Next, connect a deflated water-filled silicone balloon to a pressure transducer and a water-filled syringe using a three-way tap. After 15 to 20 minutes of an equilibration period following the previous perfusion, cut the right atrium, or RA, and insert the balloon into the right ventricle, or RV, through the RA.Use tape to hold the balloon inside the RV and minimize the open area of the RA to help constrain the balloon in the ventricle.
After 20 minutes of functional data collection for the RV, cut the left atrium, or LA, and insert a deflated water-filled balloon into the left ventricle, or LV, through the LA.Use tape to hold the balloon inside the LV.To calibrate the pressure transducer, first fill a 10 milliliter syringe with warm saline and connect the syringe to the dome through a three-way tap. Open the tap and slowly fill the dome with saline before closing all taps and removing the syringe. Then, attach the filled dome to the transducer by connecting the pressure gauge to the third end of the three-way tap.
In the recording software, from the dropdown menu of the channel connecting to the transducer, select the option of Bridge Amp. Click on 0 prior to starting the recording to set the transducer reading to zero millimeters of mercury. After several seconds of recording, slowly push the syringe and increase the pressure to 100.
Click Stop to end the recording. In the Units Conversion dialog, select the area of recording for zero millimeters of mercury and click the arrow to 1 before typing zero millimeters of mercury. Similarly, select the area of recording for 100 millimeters of mercury.
Click the arrow to 2 and type 100 millimeters of mercury. Now, click OK to calibrate the transducer. After the calibration is done, rename the channel corresponding to the pressure transducer with the balloon as Ventricle Pressure, or LV Pressure, and start recording when the heart is connected to the system.
After inserting the balloon into the ventricle, adjust the water volume in the balloon using a micrometer syringe through the three-way tap to maintain the end diastolic pressure at five to 10 millimeters of mercury. Next, rename an empty channel as dP/dt and keep the derivative source channel as ventricle pressure. This channel records the ratio of pressure change in the ventricular cavity during contraction.
Select the stable period of measure before going to setting in the blood pressure module. Then, select the LV Pressure as the input channel and click selection before pressing OK.Now, click Classifier View to remove any outlier cardiac cycle, and Table View to generate the tables for the average of maximum dP/dt and minimum dP/dt for the selected period. When the mouse heart was stored in HTK buffer for zero hours prior to perfusion, higher systolic pressure was recorded for the LV as compared to the RV.Additionally, compared to the RV, the LV also displayed significantly more muscular contraction, as well as relaxation.
However, when perfusion was performed on the heart after subjecting it to eight hours of cold storage, both the LV and RV showed a significant functional reduction as compared to the zero hour stage. The decreases were observed to be more severe in the LV as compared to the RV.The contraction and relaxation of the LV after eight-hour storage were 25.1%and 30.7%of the function corresponding to zero-hour storage, while the RV displayed 32.5%and 29.1%of function, as compared to the zero-hour baseline. Indicating primary graft dysfunction of LV after prolonged storage was more significant than that of RV.The preservation quality is largely depends on the perfusion of HTK.
It is important to make sure the perfusion is consistent to precise compare different treatments. With precise measurement of a donor heart function of preservation, this method can be easily to adapt to screen drugs that can improve donor heart function and then further validate using a heterotopic transplant model.
