The aim of the method is to evaluate the functional consequences of engineered heart tissue implantation in a small animal cryoinfarction model. The advantage of this method is that it allows for the induction of relatively reproducible myocardial infarctions with stable infarction sizes. After confirming a lack of response to toe pinch, place the anesthetized guinea pig on a 40 to 42 degrees Celsius warming platform in the supine position.
Apply eye ointment and shave and depilate the thorax. Apply 25 degrees Celsius ultrasound transducer gel to the exposed skin and place the transducer from an echocardiography system onto the thorax to acquire two-dimensional parasternal long axis views in B mode from the right neck toward the left leg at the plane of the aortic valve with a concurrent visualization of the left ventricle apex to investigate the pre-operative left ventricular function. Then turn the transducer 90 degrees to obtain a short axis B mode view at the mid papillary level.
After the last image has been obtained, transfer the guinea pig to the surgical table and tape the limbs in a spread eagle position. Disinfect the exposed skin with two sequential iodine-based and 80%ethanol scrubs and make a 1.5 centimeter vertical incision in the tracheal area. Bluntly dissect the muscles covering the trachea.
When the trachea can be observed, puncture the air tube with an 18 gauge IV cannula and insert the flexible part of the cannula as a tracheal tube. Connect the tracheal tube to an animal respirator for continuous ventilation during the procedure and count the rib spaces starting at the first intercostal space to locate the fifth intercostal space. Make a two centimeter horizontal incision on the fifth intercostal space on the left side of the guinea pig and remove the subcutaneous tissues before dissecting the muscles with an electrocautery until the intercostal muscles are reached.
Gently dissect the intercostal muscles with tweezers and small scissors until the pleural space is reached and the left lung can be visualized. Insert a retractor between the ribs and open the retractor carefully until a good view of the heart is obtained. Open the pericardium approximately one centimeter in the region of the anterior left ventricular wall and place a compress on the left lung to protect the lung from damage when inducing the cryoinjury of the left ventricle.
Next, cool the tip of an eroded metal stamp with a cross-sectional diameter of 0.5 centimeters in liquid nitrogen for three minutes before pressing the probe onto the left anterior wall of the heart for 30 seconds. Then place a 200 degrees Celsius electric soldering iron inside the stamp to warm the stamp so that the metal can be removed from the tissue. After the third stamp application, remove the retractor from the intercostal space and clamp the outflow tube of the ventilator for two seconds to inflate the lungs with maximum pressure to avoid atelectasis of the lung.
Then close the ribs with two 4-0 sutures, the muscles over the ribs with a 5-0 running suture and the skin with 5-0 suture single stitches. After removing the tracheal tube, use a single 8-0 suture to close the puncture site at the trachea and close the wound with three single stitch 5-0 sutures. Seven days after cryoinjury, remove sutures and make a two centimeter horizontal skin incision in the scar area of the left lateral side and use electrocautery to gently dissect the extra pleural adhesions.
Carefully open the pleural space with scissors and insert a rib spreader to expose the heart. Visually identify the region of the infarction by its pale color compared to the healthy surrounding myocardium and place an engineered heart tissue patch over the infarction region. Secure the patch with two 8-0 sutures at both sides in the non-infarcted area of the heart and inflate the lungs with pressure to avoid atelectasis of the lungs.
Then remove the retractor from the intercostal space and close the animal as demonstrated. Four weeks after engineered heart tissue implantation, perform transthoracic echocardiography as demonstrated to evaluate changes in the left ventricle function overtime. Mason trichrome staining four weeks after cryoinjury reveals large transmural scars over 25%of the left ventricular myocardium on average.
Dystrophin staining demonstrates large myocardial grafts that have partially re-muscularized to the scar and staining for human Ku80 that confirms the human origin of the newly formed myocardium. Transthoracic echocardiography monitoring of the implanted engineered heart tissue reveals an improvement in the left ventricle ejection fraction, fractional area shortening, and a decrease in left ventricular and diastolic diameter. It is essential to carefully open the pleural cavity in this kind of re-do operation as adhesions between the thoracic wall and the left ventricle can be present and there's a certain risk to injure the left ventricular myocardium.
After the implantation of the human engineered heart tissue, several methods can be used to monitor for changes in left ventricular ejection fraction. Besides echocardiography, an invasive measurement of pressure volume loops can be used to monitor those changes. This method allows to investigate all aspects of human engineered tissue implantation.
Functional consequences as well as potential risks like arrhythmias can be evaluated in this small animal model.
