Medicine
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A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents
Chapters
Summary September 21st, 2021
This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.
Transcript
Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves, capable of somatic growth with no requirement for anticoagulation. This technique is practical and more straightforward than previous methods for studying heart valve transplant immunobiology. After anesthetizing the rat, use the dissecting scissors to incise the skin from the xiphoid to the sternal notch.
And perform a sternectomy by cutting the ribs on each side, lateral to the sternum until optimal access to the heart is achieved. Inject Heparin in the left atrium. Post sacrification, excise the thymus to improve the visualization of the great vessels.
Then remove the heart on block with the ascending aorta till the level of the innominate artery. After placing the donor heart in a sterile Petri dish immediately following the cardiectomy, in an ice-cold, cold storage buffer, use the forceps and Vannas Spring Scissors to dissect the heart until only the aortic root remains with a one millimeter ventricular cuff proximal to the aortic valve. For opening the aortic valve, make a longitudinal cut to open the sinus of Valsalva between the left and noncoronary sinuses to visualize all three leaflets.
To excise each aortic valve leaflet individually, use the blunt forceps to grasp the edge of the leaflet and use the Vannas Spring Scissors to excise the leaflet by cutting from one commissure down to the annulus then toward the next commissure. Store the excised leaflets in ice-cold storage buffer solution until they are ready to be implanted in the recipient rat, but implant all the leaflets within four hours of cold storage. Once the recipient rat is completely anesthetized, maintain the body temperature at 36 to 38 degrees celsius throughout the surgery with the heating pad.
Next, place the rat in a right lateral recumbent position to access the left kidney. Administer Buprenorphine to recipient rat subcutaneously before surgery. And use the scissors to incise the skin over the flank longitudinally for one inch.
Similarly, incise the underlying abdominal wall. Using the thumb and forefinger, apply a light pressure dorsally and ventrally while using the curved forceps. Lift the caudal pole of the kidney through the abdominal and skin incision.
Externalize the cranial end of the kidney similarly. Alternatively, the kidney may be externalized by grasping the perirenal fat and pulling upward with light tension. Once externalized, keep the kidney moist with warm saline swabbed onto the kidney.
In order to create a subcapsular pocket, begin by applying light pressure to the renal capsule using one set of blunt forceps so that the renal capsule can be clearly distinguished from the underlying parenchyma. Simultaneously using another set of blunt forceps, carefully grasp the capsule and gently pull upward to create a hole in the capsule. With blunt forceps, extend the incision until a space of about two millimeters has been created to accommodate the aortic valve leaflet.
Develop a shallow subcapsular pocket that is slightly larger than the valve leaflet while lifting the edge of the incision with one set of forceps and advancing a blunt probe under the renal capsule. Initiate transplantation of the retrieved aortic valve by lifting the edge fibrous capsule advancing the aortic leaflet into the subcapsular pocket with blunt forceps. The incision and the renal capsule can be left open.
Push the kidney gently back into its anatomical position using countertraction applied to the incision edges. Close the abdominal incision with a running sterile surgical suture and close the skin with staples. Following the operation, place the rat in a clean cage on a heating pad with access to food and water.
If required, administer Buprenorphine to the recipient rat every six to twelve hours to alleviate the pain. Monitor the animal daily to assess for routine wound healing and signs of pain or distress. And remove the staples after seven to ten days of the surgery.
A representative image shows the position of the aortic valve leaflet under the renal capsule for implementation. And after three, seven, and twenty eight days within the recipient rat, locating and recovering the transplanted tissue became easy. The aortic valve leaflets retained their native architecture after heterotopic transplantation in syngeneic animals.
Demonstrating the utility of this model as a baseline to compare the immune response in allergenic transplants. The Histology with hematoxylin and eosin staining revealed that valve leaflets in syngeneic transplants after seven days were structurally intact with no signs of edematous swelling. The structural integrity of the valve leaflet after transplantation was further confirmed by Immunohistochemistry for smooth muscle actin and CD31.
The aortic valve leaflets are very delicate. It is important to handle them very carefully without damaging the valves. Renal subcapsular, heart valve transplantation simplifies heterotopic heart valve transplantation.
This will allow researchers with limited microsurgical capabilities to explore the immunobiology of heart valve transplantation.
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