This protocol is a large animal model for studying right ventricular adaptation and failure. This model is therefore vital for developing RV-targeted therapeutics to treat pulmonary hypertension. This model progressively increases the right RV after load which can be used as a strategy to titrate the RV adaptation.
No previous large animal models have this control over the RV phenotype. Demonstrating that surgical procedure will be Dr.Matthew Bacchetta, the laboratory director and the surgical fellows doctors John Stokes, Kelly Wu and Yatrik Patel. I will be demonstrating the pulmonary artery banding procedure.
After anesthetization, begin by shaving the surgical field from the sheep's neck to its upper abdomen. Clean the surgical field off dirt and other contaminants using soap or a scrub brush. Prep the neck in the chest with chlorhexidine or betaine solution and drape the surgical field in a sterile fashion.
To obtain media style exposure perform a muscle sparing mini thoracotomy at the left fourth intercostal space with incision length less than eight centimeters. Use a small or medium Finochietto retractor to separate the ribs in a tuffier retractor to sit perpendicular to the Finochietto within the intercostal space which will retract the soft tissue within the intercostal space, improving exposure without injuring the phrenic nerve incise the pericardia interior to it and create a pericardial well with 2/0 silk sutures to expose the main pulmonary artery and right ventricle. Identify the left atrial appendage within the exposure as a landmark for the level of the pulmonary artery bifurcation dissect around the main pulmonary artery and isolate it with a umbilical tape.
Perform intrapericardial dissection of the left pulmonary artery and encircle with a umbilical tape. Place a heavy duty silicone vascular occluder around the main pulmonary artery. Using an 0 silk suture on a Keith needle.
Secure the end to the vascular occluder together with a U-stitch. Once secured around the main pulmonary artery slide the occluder distally along the main pulmonary artery. To establish a right ventricular pressure line, start by placing a 5/0 monofilament non-absorbable polypropylene purser suture with pledgets surrounding the selected location and see a vascular snare.
Prepare the right ventricular pressure line by cutting off the male end of a sterile 36 inch pressure tubing at a 30 degree angle to facilitate insertion through the myocardium. Then use a 2/0 silk tie to mark the pressure line at an optimal depth for placement within the right ventricle. Using a biopsy punch, make a small cardiotomy in the right ventricular outflow tract or RVOT free wall within the previously placed purser string suture.
Control the bleeding with manual pressure or by tightening the snare on the purser suture. Insert and secure the cut end of the pressure tubing into the RVOT. Tie down the purse string then secure the purse string to the pressure tubing to secure the pressure line.
Extend the RVOT tubing by connecting an additional pressure tubing to the RVOT pressure line. Monitor for the measurement of the baseline, right ventricular pressure. Carefully dissect around the left pulmonary artery Encircle the left pulmonary artery with umbilical tape tying it down to ligate it.
Bring the RVOT pressure line and pulmonary artery occluder tubing out of the chest one intercostal space below the thoracotomy incision. To mark sites for indwelling ports form two subdural pockets along the fascia layer on the left dorsum of the sheep as far posteriorly toward the spine as feasible within sterile field. Use a chest tube polar to tunnel the RVOT pressure line and occluder tubing from the chest incision out to the left dorsum port sites.
Secure both the occluder tubing and right ventricular pressure line to the port's barb connections. Anchor the occluder and pressure tubing around the port connectors with additional ties. To prevent port migration anchor the ports in three locations around their rims to the underlying fascia with 3/0 polypropylene sutures.
Reapproximate the subcutaneous tissue dermis and skin in layers with polyglactin 910 sutures. Flush the RVOT port with five milliliters of heparin sodium. Close the thoracotomy with figure of eight number two polyglactin 910 sutures Then close the pectorals muscle layer with a running number 0 polyglactin 910.
Finally, close the subcutaneous tissue in layers of running number 2/0 polyglactin 910 sutures and staple the skin. Prepare two pressure transducers for monitoring, right ventricular and occluder cuff pressures. After the animal is mildly restrained insert the Huber needle from the right ventricular pressure transducer to the right ventricular port.
Attach a five milliliter syringe to the three-way stop cock and attempt to draw blood back into the syringe from the right ventricular port. Once the right ventricular pressure line is established, connect the Huber needle from the pulmonary artery cuff transducer. Capture the starting values of rip ventricular and pulmonary artery cuff pressures, noting any drastic changes from previous readings.
Slowly inject 3%hypertonic saline into the occluder port while paying attention to rip ventricular and cuff pressures. The once the PA cuff is inflated to the desired amount remove the Huber needle from the cuff port. Flush the right ventricular port with 10 milliliters of saline then flush the port with five milliliters of 1000 units per milliliter of heparin sodium.
Among the 12 sheep. The mean pulmonary artery cuff pressure increased with an increase in time from the first to the ninth week. Faster pulmonary artery banding lead to a more rapid decline in venous oxygen saturation.
In contrast, those that experienced a more gradual pulmonary artery banding strategy maintain a physiologic range of venous oxygen saturation between 70 and 80%By controlling the pulmonary artery cuff pressure. This model has ability to control and titrate ride ventricular adaptation and phenotype.
