Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

This protocol is significant because it's designed to answer questions about the structure function correlate of the murine pulmonary valve, which is generally tricky because of its inherent heterogeneity. The controlled fixation and correlative imaging were used to capture the structure on multiple length scales. Local high-resolution images can then be mapped back to the precise anatomical location on the pulmonary valve.

Demonstrating the procedure will be Dr.Tai Yi, the Director of Microsurgery in the Center of Regenerative Medicine at Nationwide Children's Hospital. Begin by autoclaving the tools needed for the mouse dissection, including fine scissors, microforceps, microvascular clamps, clamp applying forceps, microneedle holders, spring scissors, and retractors. After euthanizing an adult C57BL/6 mouse, place it in a dorsal recumbent 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 two millimeters above the ventriculoatrial 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 one millimeter distance between the sinotubular 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 three hours, refilling the reservoir to maintain a constant pressure. After perfusion, store the heart valve in the fixative solution at four degrees Celsius until use for up to one week.

For staining, wash the fixed heart valve sample three times with cold 0.15 molar cacodylate buffer for five minutes and completely submerge the heart valve in a solution of 1.5 potassium ferrocyanide, 0.15 molar cacodylate, two millimolar calcium chloride, and 2%osmium tetroxide on ice for one hour. Wash the samples with room temperature double distilled water by placing them in a tube for five minutes with slight agitation. After three more washes, place the sample in filtered thiocarbohydrazide solution at room temperature.

After 20 minutes, wash the sample three times with water as demonstrated earlier. Next, place the sample in 2%osmium tetroxide at room temperature. After 30 minutes, wash it with water three times for five minutes each and incubate the sample in 1%uranyl acetate overnight at four degrees Celsius.

Meanwhile, prepare a solution of lead nitrate. On the following day, perform the washing step three times as demonstrated earlier, then incubate the heart valve tissue in lead aspartate solution at 60 degrees Celsius for 30 minutes. Wash the samples three more times, then perform a serial dehydration treatment on the heart valve tissues with freshly prepared 20, 50, 70, and 90%ethanol on ice for five minutes each, followed by two subsequent treatments with 100%ethanol.

After dehydration, move the tissues to ice cold acetone, then place it in fresh acetone at room temperature for 10 minutes. For embedding, make the resin mixture according to the manufacturer's specifications. Place tissues in subsequent treatments of 25 to 75, 50 to 50 and 75 to 25 resin to acetone mixture for two hours each.

Finally, place the tissues in 100%resin overnight. On the following day, place tissues in fresh 100%resin. After two hours, transfer the tissues to an embedding capsule, add fresh 100%resin and place it in a 60 degree Celsius oven for 48 hours to cure.

The excised anastomosed pulmonary artery before and following the application of hydrostatic pressure is shown here. The pulmonary trunk distends radially indicating that the pulmonary valve leaflets are in a closed configuration. Microcomputed tomography confirmed the pulmonary valve confirmation.

The leaflets were closed and the annulus was circular while varying degrees of inadequate pulmonary valve pressurization by either fixation or arterial collapse were observed. The micro CT volume rendering virtual cross-sections was correlated with optical images to confirm the slicing direction and location. High-resolution SBF SEM images were taken at a local region within a leaflet when the specimen block was at the desired location and orientation.

Correlated images between the micro CT volume rendering virtual slice, low-resolution, and high-resolution SBF SEM images are shown here. In 3D representation of a segmented region of the pulmonary valve, extracellular components like endothelial cells, valvular interstitial cells, and extracellular fibers can be identified. When attempting this protocol, please keep in mind that performing the pressurization is critical and will ensure the yield to be as high as possible.