Pressure Fixation of Isolated Murine Pulmonary Valve: A Procedure to Isolate and Chemically Fix the Mouse Pulmonary Valve in Closed Conformation

To begin, secure a euthanized mouse in the supine position. Incise laterally through the thoracic wall to access the thoracic cavity and expose the heart. Excise the right atrium and inject saline into the left ventricle to drain blood from the heart.

Sever the vascular connections by cutting off the major blood vessels. Further, incise the pulmonary artery at a suitable distance from the ventriculo-arterial junction, which serves as the conduit for pressurization. Remove the ventricles to expose the ventricular side to atmospheric pressure.

Now, secure a pressurization tubing into the pulmonary artery, ensuring adequate clearance between the ventriculo-arterial junction and the tubing end to allow valve leaflets to move during pressurization.

To the tubing, connect the saline-filled reservoir fixed to a calculated height relative to the pulmonary valve based on the desired hydrostatic pressure to be applied to the arterial side of the valve. The unidirectional flow from the arterial side with the applied hydrostatic pressure results in closure of the valve leaflets and distention of the pulmonary trunk.

Subsequently, introduce a suitable fixative into the reservoir and continue the perfusion. The perfused fixative stabilizes cellular morphology and tissue architecture. 

Place a fixative-soaked gauze over the tissue to fix the tissue surrounding the valve and prevent drying. Store the fixed heart valve in fixative until use.

Begin by autoclaving the tools needed for the mouse dissection, including fine scissors, micro forceps, microvascular clamps, clamp applying forceps, microneedle holders, spring scissors, and retractors.

After euthanizing an adult C57 Black 6 mouse, place it in a dorsal recumbence position on a tray, secure its limbs with tape, and perform the thoracotomy. Expose the heart by removing any excess adipose tissue and fascia, then remove the right atrium, and perfuse the left ventricle with room temperature saline solution.

Remove the entire heart by severing the superior vena cava, inferior vena cava, pulmonary artery, and aorta, and cut approximately 2 millimeters above the ventriculo-arterial junction, which will serve as the conduit for pressurization.

Remove the left and right ventricles to expose the chambers to atmospheric pressure, ensuring that the pulmonary trunk structure is unaffected by removing the ventricles. Anastomose pressurization tubing with the pulmonary artery, leaving approximately 1-millimeter distance between the sino-tubular junction and the end of the tubing, to accommodate for large movements of the leaflets and pulmonary trunk.

Elevate the reservoir to an analogous physiological pressure and fill it with the saline solution. Test the flow-through system to ensure there are no blockages or air bubbles. Attach a stopcock to the anastomosed pulmonary valve, and ensure adequate flow through the tubing by switching the outflow tract. Once the flow is sufficient, switch the outflow to the anastomosed pulmonary valve, and ensure pressurization of the pulmonary trunk, identified by trunk distension.

After confirming the pressurization of the pulmonary trunk, gradually incorporate primary fixative solution, until 25% of the reservoir capacity of the saline solution is purged. Place a fixative-soaked gauze over the tissue sample to prevent drying. Perfuse the fixative for 3 hours, refilling the reservoir to maintain a constant pressure.

After perfusion, store the heart valve in the fixative solution at 4 degrees Celsius until use, for up to 1 week.